Recombinant vectors containing polycistronic expression cassettes and methods of use thereof

Abstract

Provided herein is a vector comprising a polycistronic expression cassette, the vector comprising a polynucleotide encoding a CD19-specific chimeric antigen receptor, a polynucleotide encoding a cytokine, and a polynucleotide encoding a marker protein, wherein the polynucleotide encoding the CD19-specific chimeric antigen receptor and the polynucleotide encoding the cytokine coding sequence are separated by a polynucleotide sequence comprising an F2A element, and the polynucleotide encoding the cytokine and the polynucleotide encoding the marker protein are separated by a polynucleotide sequence comprising a T2A element.

Description

[Technical field]

[0001] Incorporating sequence tables The sequence listing contained in the file entitled "P35188WO00_SL.TXT", which is 261,925 bytes (measured in MS-Windows®) and was created on January 7, 2022, is submitted electronically herewith and is incorporated by reference in its entirety.

[0002] The present disclosure relates to polycistronic vectors containing at least three cistrons and methods of using same. [Background technology]

[0003] Co-expression of multiple genes in each cell of a population is important for a wide variety of biomedical applications, including adoptive cell therapy, e.g., chimeric antigen receptor T cell (CAR T cell) therapy. A standard strategy for multigene expression is to incorporate transgenes into multiple vectors and introduce each vector into cells. However, the use of multiple vectors often produces a substantially heterogeneous population of engineered cells, where not all cells express each of the transgenes or do not express each of the transgenes to the same extent. Such heterogeneity causes several problems, especially for therapeutic applications, including, for example, reduced persistence of the desired engineered cell phenotype in vivo, complex manufacturing and purification requirements, and lot-to-lot variability of engineered cell products.

[0004] Given the problems associated with the use of multiple vectors to co-express multiple genes in a single cell, there is an unmet need for a single polycistronic vector that can not only express multiple transgenes in a single cell, but also express some or all of the transgenes to a similar extent across a cell population, resulting in an engineered cell population optimized for therapeutic use. Summary of the Invention

[0005] The present disclosure provides a recombinant vector comprising a polycistronic expression cassette, comprising a polynucleotide encoding an anti-CD19 chimeric antigen receptor (CAR), a polynucleotide encoding a fusion protein comprising IL-15 and IL-15Rα, and a polynucleotide encoding a marker protein, wherein the polynucleotide encoding the anti-CD19 CAR is separated from the polynucleotide encoding the fusion protein by a polynucleotide sequence comprising an F2A element, and the polynucleotide encoding the fusion protein is separated from the polynucleotide sequence encoding the marker protein by a polynucleotide sequence comprising a T2A element. Also provided are pharmaceutical compositions comprising cells, e.g., immune effector cells, engineered using the vectors described herein, and methods of treating subjects using these pharmaceutical compositions. The recombinant vectors disclosed herein are particularly useful for modifying immune effector cells (e.g., T cells) for use in adoptive cell therapy.

[0006] Thus, in one aspect, the present disclosure provides a recombinant vector comprising a polycistronic expression cassette, wherein said polycistronic expression cassette comprises, from 5' to 3', a transcriptional regulatory element operably linked to a polynucleotide comprising: a first polynucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain that specifically binds CD19, a transmembrane domain, and a cytoplasmic domain; a second polynucleotide sequence comprising an F2A element; a third polynucleotide sequence encoding a fusion protein comprising IL-15, or a functional fragment or variant thereof, and IL-15Rα, or a functional fragment or variant thereof; a fourth polynucleotide sequence comprising a T2A element; and a fifth polynucleotide sequence encoding a marker protein.

[0007] In some embodiments, the F2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 or the amino acid sequence of SEQ ID NO: 137 with one, two, or three amino acid modifications. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 141. In some embodiments, the F2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 or the amino acid sequence of SEQ ID NO: 138 with one, two, or three amino acid modifications. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 142.

[0008] In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 or the amino acid sequence of SEQ ID NO: 139 with one, two, or three amino acid modifications. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence SEQ ID NO: 143. In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 or 182 or the amino acid sequence of SEQ ID NO: 140 or 182 with one, two, or three amino acid modifications. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 144, 145, or 165.

[0009] In some embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) comprising complementarity determining regions VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising complementarity determining regions VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the antigen-binding domain comprises an scFv comprising the VH and the VL operably linked via a first peptide linker.

[0010] In some embodiments, said VH comprises the VH CDR1, VH CDR2 and VH CDR3 amino acid sequences shown in SEQ ID NO: 2. In some embodiments, said VH CDR1 comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 with one, two or three amino acid modifications, said VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 with one, two or three amino acid modifications, and said VH CDR3 comprises the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 8 with one, two or three amino acid modifications.

[0011] In some embodiments, said VL comprises the VL CDR1, VL CDR2 and VL CDR3 amino acid sequences set forth in SEQ ID NO: 1. In some embodiments, said VL CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one, two or three amino acid modifications, said VL CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one, two or three amino acid modifications, and said VL CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one, two or three amino acid modifications.

[0012] In some embodiments, the VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VH is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 20.

[0013] In some embodiments, the VL comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 19.

[0014] In some embodiments, the first peptide linker comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:17, or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:17 containing one, two, or three amino acid modifications. In some embodiments, the first peptide linker is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:27 or SEQ ID NO:35. In some embodiments, the first peptide linker is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:27.

[0015] In some embodiments, the CAR further comprises a hinge region located between the antigen binding domain and the transmembrane domain of the CAR. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 37, 38, or 39, or the amino acid sequence of SEQ ID NO: 37, 38, or 39 comprising one, two, or three amino acid modifications. In some embodiments, the hinge region is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 40, 41, or 42.

[0016] In some embodiments, the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO: 43, 44, or 45, or the amino acid sequence of SEQ ID NO: 43, 44, or 45 comprising one, two, or three amino acid modifications. In some embodiments, the transmembrane domain of the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 49, 50, 51, or 52.

[0017] In some embodiments, said hinge region and said transmembrane domain together comprise the amino acid sequence of SEQ ID NO: 46, 47, or 48, or the amino acid sequence of SEQ ID NO: 46, 47, or 48 containing one, two, or three amino acid modifications. In some embodiments, said hinge region and said transmembrane domain together are encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 53, 54, 55, or 56.

[0018] In some embodiments, the cytoplasmic domain comprises a primary signaling domain of human CD3ζ, or a functional fragment or variant thereof. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 67 or 68.

[0019] In some embodiments, the cytoplasmic domain comprises a costimulatory domain of a protein selected from the group consisting of CD28, 4-1BB, OX40, CD2, CD7, CD27, CD30, CD40, CDS, ICAM-1, LFA-1, B7-H3, and ICOS, or a functional fragment or variant thereof. In some embodiments, the protein is CD28 or 4-1BB.

[0020] In some embodiments, the protein is CD28. In some embodiments, the cytoplasmic domain comprises the amino acid sequence of SEQ ID NO: 57 or 58, or the amino acid sequence of SEQ ID NO: 57 or 58 comprising one, two, or three amino acid modifications. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 64 or 65.

[0021] In some embodiments, the protein is 4-1BB. In some embodiments, the cytoplasmic domain comprises the amino acid sequence of SEQ ID NO:59, or the amino acid sequence of SEQ ID NO:59 containing one, two, or three amino acid modifications. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:66.

[0022] In some embodiments, the cytoplasmic domain comprises the amino acid sequence of SEQ ID NO: 61, 62 or 63, or the amino acid sequence of SEQ ID NO: 61, 62 or 63 containing one, two or three amino acid modifications. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 69, 70 or 71.

[0023] In some embodiments, the CAR comprises an amino acid sequence that is at least at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72, 74, 76, 77, 78, 79, 80, or 81. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 82, 83, 86, 87, 90, 91, 92, 93, 94, or 95.

[0024] In some embodiments, said IL-15, or said functional fragment or variant thereof, is operably linked to said IL-15Rα, or said functional fragment or variant thereof, via a second peptide linker. In some embodiments, said fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 119, 121, or 180. In some embodiments, said fusion protein is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 126, 127, 130, 131, or 181.

[0025] In some embodiments, the above-mentioned marker protein comprises domain III of HER1, or a functional fragment or variant thereof, the N-terminal portion of domain IV of HER1, and the transmembrane domain of CD28, or a functional fragment or variant thereof.

[0026] In some embodiments, said domain III of HER1, or a functional fragment or variant thereof, comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, said domain III of HER1, or a functional fragment or variant thereof, is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 110 or 164.

[0027] In some embodiments, the N-terminal portion of domain IV of HER1 described above comprises amino acids 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, or 1-10 of SEQ ID NO: 99. In some embodiments, the N-terminal portion of domain IV of HER1 described above comprises amino acids 1-21 of SEQ ID NO: 99. In some embodiments, the N-terminal portion of domain IV of HER1 described above comprises the amino acid sequence of SEQ ID NO: 100, or the amino acid sequence of SEQ ID NO: 100 comprising one, two, or three amino acid modifications. In some embodiments, the N-terminal portion of domain IV of HER1 described above is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:112.

[0028] In some embodiments, said transmembrane region of CD28 comprises the amino acid sequence of SEQ ID NO: 101, or the amino acid sequence of SEQ ID NO: 101 comprising one, two or three amino acid modifications. In some embodiments, said transmembrane region of CD28 is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 113.

[0029] In some embodiments, the marker proteins comprise an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 97, 166, or 167. In some embodiments, the marker proteins are encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 107, 108, 109, 162, 173, or 174.

[0030] In some embodiments, the regulatory element comprises a promoter. In some embodiments, the promoter is a human elongation factor 1-alpha (hEF-1α) hybrid promoter. In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 146.

[0031] In some embodiments, the vector further comprises a polyA sequence 3' of the fifth polynucleotide sequence. In some embodiments, the polyA sequence comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:148.

[0032] In another aspect, the disclosure provides a recombinant vector comprising a polycistronic expression cassette, wherein said polycistronic expression cassette comprises, from 5' to 3', a transcriptional regulatory element operably linked to a polynucleotide comprising: a first polynucleotide sequence encoding a CAR comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 74; a second polynucleotide sequence comprising an F2A element; a third polynucleotide sequence encoding a fusion protein comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 119, 121, or 180; a fourth polynucleotide sequence comprising a T2A element; and a fifth polynucleotide sequence encoding a marker protein comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96 or 97.

[0033] In some embodiments, the F2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 or the amino acid sequence of SEQ ID NO: 137 with one, two, or three amino acid modifications. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 141. In some embodiments, the F2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 or the amino acid sequence of SEQ ID NO: 138 with one, two, or three amino acid modifications. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 142.

[0034] In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 or the amino acid sequence of SEQ ID NO: 139 with one, two, or three amino acid modifications. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence SEQ ID NO: 143. In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 or 182 or the amino acid sequence of SEQ ID NO: 140 or 182 with one, two, or three amino acid modifications. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 144, 145, or 165.

[0035] In another aspect, the present disclosure provides a recombinant vector comprising a polycistronic expression cassette, wherein said polycistronic expression cassette comprises a first polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical 5' to 3' to a polynucleotide sequence of SEQ ID NO: 82, 83, 86, or 87, a second polynucleotide sequence comprising an F2A element, and a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical 5' to 3' to a polynucleotide sequence of SEQ ID NO: 126, 127, 130, 131, or 181. The present invention provides a recombinant vector comprising a transcriptional regulatory element operably linked to a polynucleotide comprising: a third polynucleotide sequence that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence of SEQ ID NO: 107, 108, 109, or 162; a fourth polynucleotide sequence that comprises a T2A element; and a fifth polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence of SEQ ID NO: 107, 108, 109, or 162.

[0036] In some embodiments, the F2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 or the amino acid sequence of SEQ ID NO: 137 with one, two, or three amino acid modifications. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 141. In some embodiments, the F2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 or the amino acid sequence of SEQ ID NO: 138 with one, two, or three amino acid modifications. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 142.

[0037] In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 or the amino acid sequence of SEQ ID NO: 139 with one, two, or three amino acid modifications. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence SEQ ID NO: 143. In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 or 182 or the amino acid sequence of SEQ ID NO: 140 or 182 with one, two, or three amino acid modifications. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 144, 145, or 165.

[0038] In some embodiments of the recombinant vectors described herein, the vector further comprises a left inverted terminal repeat (ITR) and a right ITR, wherein said left ITR and said right ITR flank said polycistronic expression cassette. In some embodiments, the recombinant vector comprises, from 5' to 3', said left ITR, said transcriptional regulatory element, said first polynucleotide sequence, said second polynucleotide sequence, said third polynucleotide sequence, said fourth polynucleotide sequence, said fifth polynucleotide sequence, and said right ITR.

[0039] In another aspect, the disclosure provides a recombinant vector comprising a polycistronic expression cassette, wherein said polycistronic expression cassette comprises a transcriptional regulatory element operably linked to a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 149. In another aspect, the disclosure provides a recombinant vector comprising a polycistronic expression cassette, wherein said polycistronic expression cassette comprises a transcriptional regulatory element operably linked to a polynucleotide encoding an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 152.

[0040] In some embodiments, any of the recombinant vectors described herein further comprises a left inverted terminal repeat (ITR) and a right ITR, wherein said left ITR and said right ITR flank said polycistronic expression cassette. In some embodiments, said left ITR and said right ITR are ITRs of a DNA transposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, TcBuster transposon, and Tol2 transposon. In some embodiments, said DNA transposon is said Sleeping Beauty transposon.

[0041] In some embodiments of the recombinant vectors described herein, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments of the recombinant vectors described herein, the vector is a viral vector. In some embodiments of the recombinant vectors described herein, the vector is a polynucleotide.

[0042] In another aspect, the disclosure provides polynucleotides that encode an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:152.

[0043] In another aspect, the disclosure provides a cell population comprising a vector as described herein. In some embodiments, said vector is integrated into the genome of said cell population.

[0044] In another aspect, the disclosure provides a cell population comprising a polynucleotide encoding an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 152. In some embodiments, said polynucleotide is integrated into the genome of said cell population.

[0045] In another aspect, the disclosure provides a cell population comprising a polypeptide comprising an amino acid sequence encoded by a polynucleotide that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:152.

[0046] In some embodiments of the cell populations described herein, the cells comprise a CAR comprising the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81, a fusion protein comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 180, or 183, and a marker protein comprising the amino acid sequence of SEQ ID NO: 96, 97, 166, or 167. In some embodiments of the cell populations described herein, the cells comprise a CAR comprising the amino acid sequence of SEQ ID NO: 74, a fusion protein comprising the amino acid sequence of SEQ ID NO: 121, and a marker protein comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments of the cell populations described herein, the cells comprise a CAR comprising the amino acid sequence of SEQ ID NO: 75, a fusion protein comprising the amino acid sequence of SEQ ID NO: 122, and a marker protein comprising the amino acid sequence of SEQ ID NO: 97.

[0047] In some embodiments of the cell populations described herein, the cells are immune effector cells. In some embodiments, said immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, mast cells, and bone marrow derived phagocytes. In some embodiments, said immune effector cells are T cells. In some embodiments, the T cell population comprises alpha / beta T cells, gamma / delta T cells, or natural killer T (NK-T) cells. In some embodiments, the T cell population comprises CD4 + T cells, CD8 + T cells, or CD4 + T cells and CD8 + This includes both T cells.

[0048] In some embodiments of the cell populations described herein, the cells are ex vivo. In some embodiments of the cell populations described herein, the cells are human.

[0049] In another aspect, the disclosure provides a method of producing a population of engineered cells, comprising: introducing into a cell population a recombinant vector comprising a left ITR and a right ITR, wherein said left ITR and said right ITR flank said polycistronic expression cassette, and culturing said cell population under conditions in which said transposase integrates said polycistronic expression cassette into the genome of said cell population, thereby producing a population of engineered cells. In some embodiments, the recombinant vector comprises, from 5' to 3', said left ITR, said transcriptional regulatory element, said first polynucleotide sequence, said second polynucleotide sequence, said third polynucleotide sequence, said fourth polynucleotide sequence, said fifth polynucleotide sequence, and said right ITR.

[0050] In some embodiments, the left ITR and the right ITR are ITRs of a DNA transposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, TcBuster transposon, and Tol2 transposon. In some embodiments, the DNA transposon is the Sleeping Beauty transposon. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the Sleeping Beauty transposase is selected from the group consisting of SB11, SB100X, hSB110, and hSB81. In some embodiments, the Sleeping Beauty transposase is SB11. In some embodiments, the SB11 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, said SB11 is encoded by a polynucleotide sequence that is at least at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 161. In some embodiments, said polynucleotide encoding said DNA transposase is a DNA vector or an RNA vector.

[0051] In some embodiments, the left ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 155 or 156, and the right ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 157, 159 or 184.

[0052] In some embodiments, the recombinant vector and the DNA transposase or the polynucleotide encoding the DNA transposase are introduced into the cell population using electrotransfer, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, mechanical deformation by passage through a microfluidic device, or a colloidal dispersion system. In some embodiments, the recombinant vector and the DNA transposase or the polynucleotide encoding the DNA transposase are introduced into the cell population using electrotransfer. In some embodiments, the method is completed in less than 2 days. In some embodiments, the method is completed in 1-2 days. In some embodiments, the method is completed in more than 2 days.

[0053] In some embodiments, the cell population is cryopreserved and thawed prior to introduction of the recombinant vector and the DNA transposase or the polynucleotide encoding the DNA transposase. In some embodiments, the cell population is rested prior to introduction of the recombinant vector and the DNA transposase or the polynucleotide encoding the DNA transposase. In some embodiments, the cell population comprises human ex vivo cells. In some embodiments, the cell population is not activated ex vivo. In some embodiments, the cell population comprises T cells.

[0054] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cell population described herein, thereby treating the cancer.

[0055] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an engineered cell population produced by the methods of producing an engineered cell population described herein, thereby treating the cancer.

[0056] In another aspect, the disclosure provides a method of treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cell population described herein, thereby treating the autoimmune disease or disorder.

[0057] In another aspect, the disclosure provides a method of treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered cell population produced by the methods of producing an engineered cell population described herein, thereby treating the autoimmune disease or disorder.

[0058] In some embodiments, any of the polynucleotide sequences described herein (e.g., the polynucleotide sequences described in Tables 1-7, 10, 11, and 13) may be followed at the 3' end by a stop codon (e.g., TAA, TAG, or TGA), with or without an intervening polynucleotide sequence. [Brief description of the drawings]

[0059] [Figure 1] Figure 1A is a schematic diagram of a CD19-specific CAR CD19CAR, which incorporates, from N-terminus to C-terminus, an N-terminal signal sequence, an anti-human CD19 VL, a peptide linker, an anti-human CD19 VH, a human CD8α hinge domain, a human CD8α transmembrane (TM) domain, a human CD28 cytoplasmic domain, and a human CD3ζ cytoplasmic domain. Figure 1B is a schematic diagram of a membrane-bound IL-15 / IL-15Rα fusion protein mbIL15, which incorporates, from N-terminus to C-terminus, an N-terminal signal sequence, human IL-15, a linker peptide, and human IL-15Rα. Figure 1C is a schematic diagram of a marker protein HER1t, which incorporates, from N-terminus to C-terminus, an N-terminal signal sequence, domain III of human HER1, truncated domain IV of human HER1, a peptide linker, and a human CD28 TM domain. [Diagram 2]FIG. 1 is a schematic showing double transposition (dTp) and single transposition (sTp) approaches using the SB11 transposon / transposase system to generate CAR-T cells expressing CD19CAR, mbIL15, and HER1t. [Figure 3-1] Figures 3A-3E are graphs showing cell viability (Figure 3A), CD3 frequency (Figure 3B), CD19CAR expression (Figure 3C), mbIL15 expression (Figure 3D), and HER1t expression (Figure 3E) 1 day after electroporation of T cell enriched cryopreserved cell products from three separate donors with no plasmid (negative control), a 1:1 combination of plasmids DP1 and DP2 (dTp control), or plasmids A-F. [Figure 3-2] Figures 3A-3E are graphs showing cell viability (Figure 3A), CD3 frequency (Figure 3B), CD19CAR expression (Figure 3C), mbIL15 expression (Figure 3D), and HER1t expression (Figure 3E) 1 day after electroporation of T cell enriched cryopreserved cell products from three separate donors with no plasmid (negative control), a 1:1 combination of plasmids DP1 and DP2 (dTp control), or plasmids A-F. [Figure 4] 4A-4F are a series of two-parameter flow plots showing transgene co-expression assessed on day 1 and at the end of AaPC ​​stimulation cycles ("Stims") 1, 2, 3, and 4 for dTp control modified T cells from Donor A. The percentage of cells is shown in each quadrant. Specifically, FIG. 4A is a series of flow plots showing CD19CAR expression versus CD3 expression. FIG. 4B is a series of flow plots showing HER1t expression versus CD3 expression. FIG. 4C is a series of flow plots showing mbIL15 expression versus CD3 expression. FIG. 4D is a series of flow plots showing CD19CAR expression versus HER1t expression. FIG. 4E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. FIG. 4F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in FIG. 4D-4F show transgene expression on CD3+ gated cells. [Diagram 5] 5A-5F are a series of two-parameter flow plots showing transgene co-expression assessed at day 1 and at the end of Stim1, 2, 3, and 4 for plasmid A modified T cells from donor A. The percentage of cells is shown in each quadrant. Specifically, FIG. 5A is a series of flow plots showing CD19CAR expression versus CD3 expression. FIG. 5B is a series of flow plots showing HER1t expression versus CD3 expression. FIG. 5C is a series of flow plots showing mbIL15 expression versus CD3 expression. FIG. 5D is a series of flow plots showing CD19CAR expression versus HER1t expression. FIG. 5E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. FIG. 5F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in FIG. 5D-5F show transgene expression on CD3+ gated cells. [Figure 6] Figures 6A-6F are a series of two-parameter flow plots showing transgene co-expression assessed on day 1 and at the end of Stim1, 2, 3, and 4 for plasmid B modified T cells from donor A. The percentage of cells is shown in each quadrant. Specifically, Figure 6A is a series of flow plots showing CD19CAR expression versus CD3 expression. Figure 6B is a series of flow plots showing HER1t expression versus CD3 expression. Figure 6C is a series of flow plots showing mbIL15 expression versus CD3 expression. Figure 6D is a series of flow plots showing CD19CAR expression versus HER1t expression. Figure 6E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. Figure 6F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in Figures 6D-6F show transgene expression on CD3+ gated cells. [Figure 7]7A-7F are a series of two-parameter flow plots showing transgene co-expression assessed at day 1 and at the end of Stim1, 2, 3, and 4 for plasmid C modified T cells from donor A. The percentage of cells is shown in each quadrant. Specifically, FIG. 7A is a series of flow plots showing CD19CAR expression versus CD3 expression. FIG. 7B is a series of flow plots showing HER1t expression versus CD3 expression. FIG. 7C is a series of flow plots showing mbIL15 expression versus CD3 expression. FIG. 7D is a series of flow plots showing CD19CAR expression versus HER1t expression. FIG. 7E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. FIG. 7F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in FIG. 7D-7F show transgene expression on CD3+ gated cells. [Figure 8] 8A-8F are a series of two-parameter flow plots showing transgene co-expression assessed at day 1 and at the end of Stim1, 2, 3, and 4 for plasmid D modified T cells from donor A. The percentage of cells is shown in each quadrant. Specifically, FIG. 8A is a series of flow plots showing CD19CAR expression versus CD3 expression. FIG. 8B is a series of flow plots showing HER1t expression versus CD3 expression. FIG. 8C is a series of flow plots showing mbIL15 expression versus CD3 expression. FIG. 8D is a series of flow plots showing CD19CAR expression versus HER1t expression. FIG. 8E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. FIG. 8F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in FIG. 8D-8F show transgene expression on CD3+ gated cells. [Figure 9]9A-9F are a series of two-parameter flow plots showing transgene co-expression assessed at day 1 and at the end of Stim1, 2, 3, and 4 for plasmid E modified T cells from donor A. The percentage of cells is shown in each quadrant. Specifically, FIG. 9A is a series of flow plots showing CD19CAR expression versus CD3 expression. FIG. 9B is a series of flow plots showing HER1t expression versus CD3 expression. FIG. 9C is a series of flow plots showing mbIL15 expression versus CD3 expression. FIG. 9D is a series of flow plots showing CD19CAR expression versus HER1t expression. FIG. 9E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. FIG. 9F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in FIG. 9D-9F show transgene expression on CD3+ gated cells. [Figure 10] 10A-10F are a series of two-parameter flow plots showing transgene co-expression assessed at day 1 and at the end of Stim1, 2, 3, and 4 for plasmid F modified T cells from donor A. The percentage of cells is shown in each quadrant. Specifically, FIG. 10A is a series of flow plots showing CD19CAR expression versus CD3 expression. FIG. 10B is a series of flow plots showing HER1t expression versus CD3 expression. FIG. 10C is a series of flow plots showing mbIL15 expression versus CD3 expression. FIG. 10D is a series of flow plots showing CD19CAR expression versus HER1t expression. FIG. 10E is a series of flow plots showing CD19CAR expression versus mbIL15 expression. FIG. 10F is a series of flow plots showing HER1t expression versus mbIL15 expression. The flow plots in FIG. 10D-10F show transgene expression on CD3+ gated cells. [Figure 11-1]11A-11C are bar graphs showing transgene expression assessed on day 1 and at the end of Stim1, 2, 3, and 4 for CD3 enriched T cells from donor A transfected with dTp control or plasmids A-F. Bar graphs show expression (CD3+ gated) of CD19CAR (FIG. 11A), mbIL15 (FIG. 11B), and HER1t (FIG. 11C). [Figure 11-2] 11A-11C are bar graphs showing transgene expression assessed on day 1 and at the end of Stim1, 2, 3, and 4 for CD3 enriched T cells from donor A transfected with dTp control or plasmids A-F. Bar graphs show expression (CD3+ gated) of CD19CAR (FIG. 11A), mbIL15 (FIG. 11B), and HER1t (FIG. 11C). [Figure 12-1] Figures 12A-12C are images of Western blots confirming expression of CD19CAR (Figure 12A), mbIL15 (Figure 12B), and HER1t (Figure 12C) in cell lysates from ex vivo expanded CD19CAR-mbIL15-CAR-T cells. Cells from one normal donor are shown, except where an additional donor is indicated (in samples labeled Z). [Figure 12-2] Figures 12A-12C are images of Western blots confirming expression of CD19CAR (Figure 12A), mbIL15 (Figure 12B), and HER1t (Figure 12C) in cell lysates from ex vivo expanded CD19CAR-mbIL15-CAR-T cells. Cells from one normal donor are shown, except where an additional donor is indicated (in samples labeled Z). [Figure 13] 13A-13C are graphs showing estimated cell numbers assessed at day 1 and at the end of Stim1, 2, 3, and 4 for T cell enriched starting products from donor A transfected and ex vivo expanded with dTp control or plasmids A-F. Estimated cell numbers for CD3 gated CD19CAR+ (FIG. 13A), mbIL15+ (FIG. 13B), and HER1t+ (FIG. 13C) were plotted over time. [Figure 14-1]14A-14H are graphs showing the cytotoxicity of ex vivo expanded CD19-specific T cells either untransfected (negative control) (FIG. 14A) or transfected with dTp control (FIG. 14B), Plasmid A (FIG. 14C), Plasmid B (FIG. 14D), Plasmid C (FIG. 14E), Plasmid D (FIG. 14F), Plasmid E (FIG. 14G), and Plasmid F (FIG. 14H) as determined by chromium release assay against CD19+ (Daudi β2M, NALM-6, and CD19 EL-4) and CD19-negative (parental EL-4) target cells at different effector-to-target (E:T) ratios by measuring the lysis of radiolabeled (51Cr) target cells. The mean ± standard deviation (SD) percent lysis of several E:T triplicate wells is shown for cells derived from donor A. Error bars represent SD and may be obscured by the symbols. [Figure 14-2] 14A-14H are graphs showing the cytotoxicity of ex vivo expanded CD19-specific T cells either untransfected (negative control) (FIG. 14A) or transfected with dTp control (FIG. 14B), Plasmid A (FIG. 14C), Plasmid B (FIG. 14D), Plasmid C (FIG. 14E), Plasmid D (FIG. 14F), Plasmid E (FIG. 14G), and Plasmid F (FIG. 14H) as determined by chromium release assay against CD19+ (Daudi β2M, NALM-6, and CD19 EL-4) and CD19-negative (parental EL-4) target cells at different effector-to-target (E:T) ratios by measuring the lysis of radiolabeled (51Cr) target cells. The mean ± standard deviation (SD) percent lysis of several E:T triplicate wells is shown for cells derived from donor A. Error bars represent SD and may be obscured by the symbols. [Figure 15]Graph showing antibody-dependent cellular cytotoxicity (ADCC) of ex vivo expanded CD19CAR-mbIL15-HER1t T cells. Genetically modified T cells served as targets in a chromium release assay in the presence of cetuximab (EGFR-specific antibody) or rituximab (CD20-specific antibody, negative control) using Fc receptor-expressing NK cells as effectors. Mock-transfected (no DNA) T cells were used as negative control. Data for donor A at an E:T ratio of 40:1 are shown. Bar graphs represent the mean lysis values ​​of genetically modified T cells normalized to the maximum NK cell lysis percentage. [Figure 16] Graph showing transgene copy number in ex vivo expanded CD19CAR-mbIL15-HER1t T cells from donor A transfected with double transposon control or test plasmids (dTp control or plasmids A-F, respectively), mock transfected CD3 (negative control with no DNA), CD19CAR+Jurkat cells (positive control for CD19CAR), mbIL15+Jurkat cells (positive control for mbIL15), or CD19CAR+HER1t+ T cells (positive control for HER1t). Copy numbers were assessed in quintuplicate for each sample using ddPCR and normalized to the human reference gene EIF2C1. [Figure 17] 17A-17C are graphs showing estimated cell numbers assessed at day 1 and at the end of Stim1, 2, 3, and 4 for dTp control (FIG. 17A), Plasmid A (FIG. 17B), and Plasmid D (FIG. 17C) electroporated T cell enrichment products expanded ex vivo by co-culture on irradiated clone 9 AaPCs. Total cells, CD3+, CD3+-gated CD19CAR+, and CD3+-gated HER1t+ expansion over time are plotted and shown as mean ± SD of multiple donor samples pooled from multiple experiments. Error bars represent SD and may be obscured by the symbols. [Figure 18-1]Figures 18A-18C are bar graphs showing percent transgene subpopulation heterogeneity (CD19CAR+HER1t-negative, CD19CAR+HER1t+, CD19CAR-negative HER1t+, CD19CAR-negative HER1t-negative) plotted at 18 hours (day 1) and Stims4 post-electroporation for dTp control (Figure 18A), Plasmid A (Figure 18B), and Plasmid D (Figure 18C) T cell enriched products expanded ex vivo by co-culture on irradiated Clone 9 AaPCs. Data are shown as mean ± SD of multiple donor samples pooled from multiple experiments. [Figure 18-2] Figures 18A-18C are bar graphs showing percent transgene subpopulation heterogeneity (CD19CAR+HER1t-negative, CD19CAR+HER1t+, CD19CAR-negative HER1t+, CD19CAR-negative HER1t-negative) plotted at 18 hours (day 1) and Stims4 post-electroporation for dTp control (Figure 18A), Plasmid A (Figure 18B), and Plasmid D (Figure 18C) T cell enriched products expanded ex vivo by co-culture on irradiated Clone 9 AaPCs. Data are shown as mean ± SD of multiple donor samples pooled from multiple experiments. [Figure 19] 19A-19C are graphs showing the cytotoxicity of ex vivo expanded CD19-specific T cells transfected with dTp control (FIG. 19A), Plasmid A (FIG. 19B), or Plasmid D (FIG. 19C) as determined by chromium release assay against CD19+ (Daudi β2M, NALM-6, and CD19 EL-4) and CD19-negative (parental EL-4) target cells at different effector-to-target (E:T) ratios by measuring the lysis of radiolabeled (51Cr) target cells. Mean ± SD percent lysis is shown for multiple donors pooled from multiple experiments. Error bars represent SD and may be obscured by the symbols. [Figure 20]Graph showing antibody-dependent cellular cytotoxicity (ADCC) of ex vivo expanded CD19CAR-mbIL15-HER1t T cells. Genetically modified T cells served as targets in a chromium release assay in the presence of cetuximab (EGFR-specific antibody) or rituximab (CD20-specific antibody, negative control) using Fc receptor-expressing NK cells as effectors. Mean ± SD percent lysis at an E:T ratio of 40:1 is shown for multiple donors pooled from multiple experiments. Bar graphs represent the mean lysis values ​​of genetically modified T cells normalized to maximum percent NK cell lysis. [Figure 21] Graph showing transgene copy number of ex vivo expanded CD19CAR-mbIL15-HER1t T cells transfected with dTp Control, Plasmid A, or Plasmid D, or transfected CD3 (no DNA negative control). Copy number was assessed in triplicate for each sample using ddPCR and normalized to the human reference gene EIF2C1. Data are shown as mean ± SD transgene copies per cell and represent multiple donor samples pooled from multiple experiments. [Figure 22] Figures 22A-22C are a series of two-parameter flow plots showing transgene co-expression as assessed for mock PBMC, dTp control (P, 5e6), Plasmid A (P, 5e6), and Plasmid A (T, 1e6) / Plasmid A (T, 0.5e6), as defined in Example 4. The percentage of cells is shown in each quadrant. Specifically, Figure 22A is a series of flow plots showing CD19CAR expression versus CD3 expression. Figure 22B is a series of flow plots showing CD19CAR expression versus HER1t expression. Figure 22C is a series of flow plots showing HER1t expression versus mbIL15 expression. Gating strategy: lymphocytes>singlets>viable>CD3+ events. [Diagram 23]Figures 23A-23C are a series of two-parameter flow plots showing transgene co-expression as assessed for cells obtained from ex vivo expansion of mock PBMC, dTp control (P, 5e6), and Plasmid A (P, 5e6). Percentages of cells are shown in each quadrant. Specifically, Figure 23A is a series of flow plots showing CD19CAR expression versus CD3 expression. Figure 23B is a series of flow plots showing CD19CAR expression versus HER1t expression. Figure 23C is a series of flow plots showing HER1t expression versus mbIL15 expression. Gating strategy: lymphocytes>singlets>viable>CD3+ events. [Figure 24-1] 24A-24G are graphs showing tumor flux over time for NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice that were intravenously injected with 1.5×104 CD19+NALM-6 leukemia cells expressing firefly luciferase (fLUC) and then either left untreated (tumor only, FIG. 24A) or treated on day 7 with mock PBMCs (FIG. 24B), mock CD3 (FIG. 24C), dTp control (P, 5e6) (FIG. 24D), plasmid A (P, 5e6) (FIG. 24E), plasmid A (T, 1e6) (FIG. 24F), or plasmid A (T, 0.5e6) (FIG. 24G) RPM T cells. Tumor flux over time is presented for each treatment group, with each line representing an individual animal. The dotted line represents the "2x background" threshold for determining disease-free mice. [Figure 24-2]24A-24G are graphs showing tumor flux over time for NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice that were intravenously injected with 1.5×104 CD19+NALM-6 leukemia cells expressing firefly luciferase (fLUC) and then either left untreated (tumor only, FIG. 24A) or treated on day 7 with mock PBMCs (FIG. 24B), mock CD3 (FIG. 24C), dTp control (P, 5e6) (FIG. 24D), plasmid A (P, 5e6) (FIG. 24E), plasmid A (T, 1e6) (FIG. 24F), or plasmid A (T, 0.5e6) (FIG. 24G) RPM T cells. Tumor flux over time is presented for each treatment group, with each line representing an individual animal. The dotted line represents the "2x background" threshold for determining disease-free mice. [Diagram 25] Scatter plots showing tumor flux of individual mice at the last BLI before death or euthanasia. Bars represent geometric mean and SD, significance was determined by one-way ANOVA (Dunnett's post-hoc test). Error bars represent SD and may be hidden by the symbols. [Figure 26] Figures 26A-26C are Kaplan-Meier survival curves showing overall survival (OS) for each mouse treatment group. Specifically, Figure 26A is the survival curve for the tumor only treatment group (Group A). ​​Figure 26B is the survival curve for the Mock PBMC (Group B), dTp control (Group D), and Plasmid A (P, 5e6) (Group E) treatment groups. Figure 26C is the survival curve for the Mock CD3 (Group C), Plasmid A (T, 1e6) (Group F), and Plasmid A (T, 0.5e6) (Group G) treatment groups. [Figure 27]Figures 27A-27C are Kaplan-Meier survival curves showing xGvHD-free survival for each mouse treatment group. xGvHD-free survival analysis censored mice that died at low tumor burden (i.e., total flux <1x108 p / s), where mortality was likely due to xGvHD. Specifically, Figure 27A is the survival curve for the tumor-only treatment group (Group A). ​​Figure 27B is the survival curve for the Mock PBMC (Group B), dTp control (Group D), and Plasmid A (P, 5e6) (Group E) treatment groups. Figure 27C is the survival curve for the Mock CD3 (Group C), Plasmid A (T, 1e6) (Group F), and Plasmid A (T, 0.5e6) (Group G) treatment groups. [Figure 28] 28A-28C are bar graphs showing CD3+ frequency as a percentage of viable CD45+ cells in peripheral blood (PB) (FIG. 28A), bone marrow (BM) (FIG. 28B), and spleen (FIG. 28C) for each of the tumor only (group A), mock PBMC (group B), mock CD3 (group C), dTp control (group D), plasmid A (P, 5e6) (group E), plasmid A (T, 1e6) (group F), and plasmid A (T, 0.5e6) (group G) treatment groups. Cells were co-stained with antibodies including anti-CD45 and anti-CD3, followed by flow cytometry analysis. Circles represent individual mice and bars show the mean and range. [Figure 29-1] Figure 29A is a series of representative two-parameter flow plots showing expression of CD19CAR versus CD3 in cells from peripheral blood of moribund mice or mice at the end of the study in each of the seven treatment groups. Cells were co-stained with antibodies including anti-CD3, anti-CD19CAR, anti-HER1t, and anti-IL-15, followed by flow cytometry analysis. Flow plots were gated on singlets, viable hCD45+, and CD3+ events, and the frequency of each transgene was analyzed. The percentage of cells is shown for each gate. [Figure 29-2]Figures 29B-29D are bar graphs showing CD19CAR+CD3+ frequency as a percentage of viable CD45+CD3+ cells in peripheral blood (PB) (Figure 29B), bone marrow (BM) (Figure 29C), and spleen (Figure 29D) for each of the Mock PBMC (Group B), Mock CD3 (Group C), dTp control (Group D), Plasmid A (P, 5e6) (Group E), Plasmid A (T, 1e6) (Group F), and Plasmid A (T, 0.5e6) (Group G) treatment groups. Due to the lack of CD3 engraftment in tumor-only (Group A) mice, this group was excluded from presentation. Circles represent individual mice and bars show the mean and range. Error bars represent SD and may be obscured by symbols. [Diagram 30] A series of representative two-parameter flow plots showing expression of CD19CAR versus HER1t in cells from peripheral blood of moribund mice or mice at the end of the study in each of the seven treatment groups. Cells were co-stained with antibodies including anti-CD3, anti-CD19CAR, anti-HER1t, and anti-IL-15, followed by flow cytometry analysis. The displayed flow plots were gated on singlets, viable hCD45+, and CD3+ events. The percentage of cells is shown in each quadrant. [Diagram 31] A series of representative two-parameter flow plots showing expression of HER1t versus mbIL15 in cells from peripheral blood of moribund mice or mice at the end of the study in each of the seven treatment groups. Cells were co-stained with antibodies including anti-CD3, anti-CD19CAR, anti-HER1t, and anti-IL-15, followed by flow cytometry analysis. The displayed flow plots were gated on singlets, viable hCD45+, and CD3+ events. The percentage of cells is shown in each quadrant. [Diagram 32]Figures 32A and 32B are a series of representative two-parameter flow plots showing expression of CD45RO versus CCR7 (Figure 32A) or CD45RO versus CD27 (Figure 32B) in cells from peripheral blood of moribund mice or mice at the end of the study in each of the dTp control (P, 5e6) (group D), Plasmid A (P, 5e6) (group E), Plasmid A (T, 1e6) (group F), and Plasmid A (T, 0.5e6) (group G) treatment groups. Cells were co-stained with antibodies including anti-CD3, anti-CD19CAR, anti-CD45RO, anti-CCR7, and anti-CD27, followed by flow cytometry analysis. The displayed flow plots were gated on singlets, viable hCD45+, and CD3+CD19CAR+ events. [Diagram 33] Figures 33A and 33B are bar graphs representing the data shown in Figures 32A and 32B, respectively. The circles represent individual mice, the floating bars indicate minimum and maximum values, and the line represents the average. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] The present disclosure provides a recombinant polycistronic nucleic acid vector comprising at least three cistrons, from 5' to 3', a first cistron encoding an anti-CD19 chimeric antigen receptor (CAR) (e.g., CD19CAR), a second cistron encoding a fusion protein comprising IL-15 and IL-15Rα (e.g., mbIL15), or a functional fragment or variant thereof, and a third cistron encoding a marker protein (e.g., HER1t), wherein the first and second cistrons are separated by a polynucleotide sequence comprising an F2A element, and the second and third cistrons are separated by a polynucleotide sequence comprising a T2A element. Also provided are immune effector cells comprising these vectors, immune effector cells engineered ex vivo using the vectors to express the three proteins encoded by the vectors, pharmaceutical compositions comprising these vectors or engineered immune effector cells made using these vectors, and methods of treating subjects using these vectors or engineered immune effector cells made using these vectors.

[0061] The polycistronic vectors described herein are particularly useful in methods of producing populations of engineered cells (e.g., immune effector cells) that are substantially homogeneous compared to prior art systems that utilized at least two vectors for the expression of three proteins. Surprisingly, it has been further shown that the 5' to 3' order of the cistron, i.e., 5'-anti-CD19 CAR-F2A element-IL-15 / IL-15Rα fusion-T2A element-marker protein-3', provides superior expression of the three protein-encoding polynucleotide sequences, i.e., anti-CD19 CAR, IL-15 / IL-15Rα fusion, and marker protein, on the surface of T cells compared to alternative orientations.

[0062] 5.1 Definition 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 the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not intended to limit any claimed subject matter. In this application, the use of the singular includes the plural unless otherwise noted. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the term "including," as well as other forms of "include," "includes," and "included," are not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0063] As used herein, when used to modify a numerical value or numerical range, the terms "about" and "approximately" indicate a deviation of 5%-10% above (e.g., up to 5%-10% above) and 5%-10% below (e.g., up to 5%-10% below) that value or range while remaining within the intended meaning of the cited value or range.

[0064] As used herein, the term "cistron" refers to a polynucleotide sequence from which a transgene product can be produced.

[0065] As used herein, the term "polycistronic vector" refers to a polynucleotide vector that comprises a polycistronic expression cassette.

[0066] As used herein, the term "polycistronic expression cassette" refers to a polynucleotide sequence in which expression of three or more transgenes is regulated by a common transcriptional regulatory element (e.g., a common promoter) and capable of simultaneously expressing three or more separate proteins from the same mRNA. Exemplary polycistronic vectors include, but are not limited to, tricistronic vectors (containing three cistrons) and tetracistronic vectors (containing four cistrons).

[0067] As used herein, the term "transcriptional regulatory element" refers to a polynucleotide sequence that mediates the regulation of transcription of another polynucleotide sequence. Exemplary transcriptional regulatory elements include, but are not limited to, promoters and enhancers.

[0068] As used herein, the term "F2A element" refers to a polynucleotide that (i) comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 141 or 142, (ii) encodes the amino acid sequence of SEQ ID NO: 137 or 138, or (iii) encodes the amino acid sequence of SEQ ID NO: 137 or 138 containing one, two, or three amino acid modifications. In some embodiments, when located in a vector between a first polynucleotide sequence encoding a first protein and a second polynucleotide sequence encoding a second protein, the F2A element can mediate translation of the first and second polynucleotide sequences as two separate polypeptides from the same mRNA molecule, e.g., by preventing synthesis of a peptide bond between the penultimate (e.g., glycine) and last (e.g., proline) residues at the C-terminus of the translation product of the F2A element, e.g., such that the penultimate (e.g., glycine) residue becomes the C-terminal residue of the first protein and the last (e.g., proline) residue becomes the N-terminal residue of the second protein. In some embodiments, the F2A element further comprises a polynucleotide sequence at its 5' end encoding a furin cleavage site, e.g., RAKR (SEQ ID NO: 187).

[0069] As used herein, the term "T2A element" refers to a polynucleotide that (i) comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 143, 144, 145 or 165, (ii) encodes the amino acid sequence of SEQ ID NO: 139, 140 or 182, or (iii) encodes the amino acid sequence of SEQ ID NO: 139, 140 or 182 containing one, two, or three amino acid modifications. In some embodiments, when located in a vector between a first polynucleotide sequence encoding a first protein and a second polynucleotide sequence encoding a second protein, the T2A element can mediate translation of the first and second polynucleotide sequences as two separate polypeptides from the same mRNA molecule, e.g., by preventing synthesis of a peptide bond between the penultimate (e.g., glycine) and last (e.g., proline) residues at the C-terminus of the translation product of the T2A element, e.g., such that the penultimate (e.g., glycine) becomes the C-terminal residue of the first protein and the last (e.g., proline) becomes the N-terminal residue of the second protein. In some embodiments, the T2A element further comprises a polynucleotide sequence at its 5' end encoding a furin cleavage site, e.g., RAKR (SEQ ID NO: 187).

[0070] As used herein, the terms "inverted terminal repeat," "ITR," "inverted repeat / direct repeat," and "IR / DR" are used interchangeably and refer to a sequence of repeats, e.g., corresponding to, e.g., reverse complementary (e.g., perfect or incomplete) sequences, having, e.g., about 230 nucleotides (e.g., 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, or 240 nucleotides), adjacent to (e.g., with or without intervening polynucleotide sequences) opposite ends of an expression cassette (e.g., a polycistronic expression cassette). When used in combination with a polynucleotide sequence (fully reverse complementary to the transposase polypeptide), it refers to a polynucleotide sequence having about 230 nucleotides (e.g., 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, or 240 nucleotides) and adjacent to one end (e.g., with or without intervening polynucleotide sequences) of an expression cassette (e.g., a polycistronic expression cassette) cleavable by a transposase polypeptide (e.g., as described in Cui et al., J. Mol. Biol. 2002;318(5):1221-35, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the ITR, e.g., DNA transposon (e.g., Sleeping Beauty transposon, piggyBac transposon, TcBuster transposon, and Tol2 transposon), contains, e.g., two direct repeats ("DR"), e.g., imperfect direct repeats of about 30 nucleotides (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides), located at each end of the ITR. The terms "ITR" and "DR" refer to the DNA sequence of the sense strand when used in reference to a single-stranded or double-stranded DNA vector. The transposase polypeptide can recognize the sense and / or antisense strand of DNA.

[0071] As used herein, the term "left ITR" when used in reference to a linear single-stranded or double-stranded DNA vector refers to an ITR located 5' of a polycistronic expression cassette. As used herein, the term "right ITR" when used in reference to a linear single-stranded or double-stranded DNA vector refers to an ITR located 3' of a polycistronic expression cassette. When a circular vector is used, the left ITR is closer to the 5' end of the polycistronic expression cassette than the right ITR, and the right ITR is closer to the 3' end of the polycistronic expression cassette than the left ITR.

[0072] As used herein, the term "operably linked" refers to the linkage of polynucleotide or amino acid sequence elements in a functional relationship. For example, a polynucleotide sequence is operably linked when it is placed into a functional relationship with another polynucleotide sequence. In some embodiments, a transcriptional regulatory polynucleotide sequence, such as a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide sequence encoding a protein if it affects the transcription of the polynucleotide sequence encoding the protein.

[0073] The term "polynucleotide" as used herein refers to a polymer of DNA or RNA. Polynucleotide sequences may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide bonds, such as phosphoramidate or phosphorothioate bonds, instead of the phosphodiesters found between the nucleotides of unmodified polynucleotide sequences. Polynucleotide sequences include, but are not limited to, all polynucleotide sequences obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning polynucleotide sequences from recombinant libraries or cell genomes, using conventional cloning techniques and the polymerase chain reaction, and synthetic means.

[0074] The terms "amino acid sequence" and "polypeptide", used interchangeably herein, refer to a polymer of amino acids joined by one or more peptide bonds.

[0075] The term "functional variant" as used herein with reference to a protein or polypeptide refers to a protein that contains at least one amino acid modification (e.g., substitution, deletion, addition) that retains at least one specific function compared to the amino acid sequence of a reference protein. In some embodiments, the reference protein is a wild-type protein. For example, a functional variant of an IL-2 protein may refer to an IL-2 protein that contains an amino acid substitution that retains the ability to bind to an intermediate affinity IL-2 receptor but loses the ability of the protein to bind to a high affinity IL-2 receptor compared to the wild-type IL-2 protein. Not all functions of the reference wild-type protein need be retained by a functional variant of a protein. In some cases, one or more functions are selectively reduced or eliminated.

[0076] As used herein with reference to a protein or polypeptide, the term "functional fragment" refers to a fragment of a reference protein that retains at least one specific function. For example, a functional fragment of an anti-HER2 antibody may refer to a fragment of an anti-HER2 antibody that retains the ability to specifically bind to a HER2 antigen. Not all functions of the reference protein need be retained by a functional fragment of a protein. In some cases, one or more functions are selectively reduced or eliminated.

[0077] As used herein, with reference to a polynucleotide sequence, the term "modified" refers to a polynucleotide sequence that contains at least one substitution, alteration, inversion, addition, or deletion of a nucleotide compared to a reference polynucleotide sequence. As used herein, with reference to an amino acid sequence, the term "modified" refers to an amino acid sequence that contains at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference amino acid sequence.

[0078] As used herein, with reference to a polynucleotide sequence, the term "derived from" refers to a polynucleotide sequence that has at least 85% sequence identity to the reference naturally occurring nucleic acid sequence from which it is derived. With reference to an amino acid sequence, the term "derived from" refers to an amino acid sequence that has at least 85% sequence identity to the reference naturally occurring amino acid sequence from which it is derived. As used herein, the term "derived from" does not refer to any particular process or method for obtaining a polynucleotide or amino acid sequence. For example, a polynucleotide or amino acid sequence can be chemically synthesized.

[0079] As used herein, the terms "antibody" and "antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), as well as antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a , or IgG2b ). In certain embodiments, the antibodies described herein are IgG antibodies, or classes thereof (e.g., human IgG1 or IgG4), or subclasses thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0080] As used herein, the terms "VH region" and "VL region" refer to a single antibody heavy and light chain variable region comprising FR (framework regions) 1, 2, 3 and 4, and CDR (complementarity determining regions) 1, 2 and 3, respectively (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety).

[0081] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977), and Kabat et al., Sequences of proteins of immunological interest. (1991), all of which are incorporated by reference in their entirety. Unless otherwise indicated, the term "CDR" refers to the CDR as defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977), and Kabat et al., Sequences of proteins of immunological interest. (1991).

[0082] As used herein, the term "framework (FR) amino acid residues" refers to those amino acids within the framework region of an antibody variable region. The term "framework region" or "FR region" as used herein includes amino acid residues that are part of the variable region but are not part of the CDRs (e.g., using the Kabat definition of CDRs).

[0083] As used herein, the term "variable region" refers to a portion of an antibody, generally a portion of a light or heavy chain, typically approximately the amino-terminal 110-120 or 110-125 amino acids in mature heavy chains and approximately 90-115 amino acids in mature light chains, that varies significantly in sequence between antibodies and is used in the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0084] The terms "VL" and "VL domain" are used interchangeably and refer to the light chain variable region of an antibody.

[0085] The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of an antibody heavy chain.

[0086] As used herein, the terms "constant region" and "constant domain" are interchangeable and are common in the art. The constant region is the antibody portion, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors). The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to the immunoglobulin variable domain.

[0087] As used herein, the term "heavy chain", when used in reference to an antibody, may refer to any of the different types, based on the amino acid sequence of the constant domain, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0088] As used herein, the term "light chain", when used in reference to an antibody, can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0089] As used herein, the term "EU numbering system" refers to the EU numbering rules for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and Kabat et al, Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0090] As used herein, the term "specifically binds" refers to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, BIAcore®, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In specific embodiments, a molecule that specifically binds to an antigen has a K that is lower than the K that would be expected if the molecule were to non-specifically bind to another antigen. A At least 2 logs (e.g., 10 times), 2.5 logs, 3 logs, 4 logs, or more greater than K A One of skill in the art will appreciate that the antibodies described herein can specifically bind to more than one antigen (e.g., via different regions of the antibody molecule).

[0091] As used herein, the term "linked to" refers to a covalent or non-covalent bond between two molecules or moieties. One of skill in the art will understand that when a first molecule or moiety is linked to a second molecule or moiety, the linkage need not be direct, but may instead be through an intervening molecule or moiety. For example, when a heavy chain variable region of a full-length antibody is linked to a ligand binding moiety, the ligand binding moiety can be linked (e.g., via a peptide bond) to a constant region of the full-length antibody (e.g., a heavy chain constant region) rather than directly to the heavy chain variable region.

[0092] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a transmembrane protein that includes an antigen-binding domain operably linked to a transmembrane domain, which is operably linked to a cytoplasmic domain that includes at least one intracellular signaling domain. CARs can be expressed on the surface of host cells (e.g., immune effector cells) to mediate activation upon binding to a target antigen in vivo. In some embodiments, CARs specifically bind to CD19. In some embodiments, CARs specifically bind to human CD19 (hCD19).

[0093] As used herein, the term "CD19" (also known as B-lymphocyte antigen CD19, cluster of differentiation 19, and B-lymphocyte surface antigen B4) refers to the protein encoded by the CD19 gene in humans. As used herein, the term "human CD19" or hCD19 refers to the CD19 protein encoded by the human CD19 gene (e.g., wild-type human CD19 gene). Exemplary wild-type human CD19 proteins are provided by GenBank™ Accession Nos. AAB60697.1, AAA69966.1, and BAB60954.1.

[0094] As used herein, the term "extracellular" refers to one or more portions of a transmembrane protein that are located outside a cell. In some embodiments, the transmembrane protein is a recombinant transmembrane protein. In some embodiments, the recombinant transmembrane protein is a CAR.

[0095] As used herein, the term "antigen binding domain" with respect to a CAR refers to a domain of a CAR that comprises any suitable antibody or non-antibody based molecule that specifically binds to an antigen. In some embodiments, the antigen is expressed on the surface of a cell. In some embodiments, the antigen is CD19. In some embodiments, the antigen is hCD19. In some embodiments, the antibody-based molecule comprises a single chain variable fragment (scFv).

[0096] As used herein, the term "extracellular antigen binding domain" in reference to a CAR refers to an antigen binding domain that is located outside the cell. In some embodiments, the antigen binding domain is operably linked to a transmembrane domain that is operably linked to a cytoplasmic domain that includes at least one intracellular signaling domain and the antigen binding domain that is oriented so as to be located outside the cell in which the CAR is expressed.

[0097] As used herein, the term "transmembrane domain" with respect to a CAR refers to one or more portions of a CAR that are embedded in the plasma membrane of a cell when the CAR is expressed in the cell.

[0098] As used herein, the term "cytoplasmic domain" with respect to a CAR refers to one or more portions of a CAR that are located within the cytoplasm of a cell when the CAR is expressed in the cell.

[0099] As used herein, the term "intracellular signaling domain" refers to a portion of the cytoplasmic domain of a CAR that includes the primary signaling domain and / or the costimulatory domain.

[0100] As used herein, the term "primary signaling domain" refers to the intracellular portion of a signaling molecule that is responsible for mediating an intracellular signaling event.

[0101] As used herein, the term "costimulatory domain" refers to the intracellular portion of a costimulatory molecule that is involved in mediating intracellular signaling events.

[0102] As used herein, the term "cytokine" refers to a molecule that mediates and / or regulates a biological or cellular function or process (e.g., immunity, inflammation, and hematopoiesis). As used herein, cytokines include, but are not limited to, lymphokines, chemokines, monokines, and interleukins. As used herein, the term cytokine also encompasses functional variants and functional variants of wild-type cytokines.

[0103] As used herein, the term "marker" protein or polypeptide refers to a protein or polypeptide that can be expressed on the surface of a cell and can be used to mark or deplete cells that express the marker protein or polypeptide. In some embodiments, depletion of cells that express the marker protein or polypeptide is achieved by administration of a molecule that specifically binds to the marker protein or polypeptide (e.g., an antibody that mediates antibody-mediated cytotoxicity).

[0104] As used herein, "immune effector cells" refers to cells involved in promoting immune effector function. Examples of immune effector cells include, but are not limited to, T cells (e.g., alpha / beta T cells and gamma / delta T cells, CD4 + T cells, CD8 + These include T cells, natural killer T (NK-T) cells, natural killer (NK) cells, B cells, mast cells, and bone marrow-derived phagocytes.

[0105] As used herein, the term "immune effector function" refers to a specialized function of an immune effector cell. The effector function of any given immune effector cell may be different. For example, the effector function of a CD8+ T cell is cytolytic activity, and the effector function of a CD4+ T cell is secretion of cytokines.

[0106] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic or prophylactic measures as described herein. Methods of "treatment" use administering a recombinant vector comprising a polycistronic expression cassette to a cell, and in some embodiments administering the engineered cell to a subject having or previously diagnosed with a disease or disorder, to prevent, cure, delay, reduce the severity of, or ameliorate a disease or disorder, or one or more symptoms of a recurrent disease or disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0107] As used herein, in the context of administering a therapy, the term "effective amount" refers to the amount of the therapy that achieves a desired prophylactic or therapeutic effect.

[0108] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0109] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which are incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100, word length=12, etc., to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed using the XBLAST program parameters set, for example, score 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, the entire contents of which are incorporated herein by reference. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0110] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0111] 5.2 Chimeric Antigen Receptors (CARs) CAR is a transmembrane protein that comprises an antigen-binding domain operably linked to a transmembrane domain, which is operably linked to a cytoplasmic domain that comprises at least one intracellular signaling domain. CAR can be expressed on the surface of a host cell (e.g., immune effector cell) to mediate activation when it binds to a target antigen in vivo. In some embodiments, CAR specifically binds to CD19. In some embodiments, CAR specifically binds to human CD19 (hCD19).

[0112] 5.2.1 hCD19-binding domain The hCD19 binding domain comprises any suitable antibody or non-antibody based molecule that specifically binds to hCD19 expressed on the surface of a cell. Exemplary hCD19 binding domains include, but are not limited to, antibodies, as well as functional fragments and variants thereof. In some embodiments, the hCD19 binding domain comprises a single chain variable fragment (scFv), Fab, F(ab')2, Fv, full length antibody, diabody, or adnectin. In some embodiments, the hCD19 binding domain comprises an scFv.

[0113] In some embodiments, the hCD19 binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the hCD19 binding domain comprises a VH and a VL operably linked via a peptide linker. In some embodiments, the peptide linker comprises glycine (G) and serine (S).

[0114] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO:9.

[0115] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO: 17.

[0116] In some embodiments, the linker is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide of SEQ ID NO: 27. In some embodiments, the linker is encoded by the polynucleotide of SEQ ID NO: 27.

[0117] In some embodiments, the linker is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide of SEQ ID NO: 35. In some embodiments, the linker is encoded by the polynucleotide of SEQ ID NO:35.

[0118] In some embodiments, the VH comprises three complementarity determining regions (CDRs): VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 as set forth in SEQ ID NO:2. In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence containing one, two, or three amino acid modifications relative to the amino acid sequence of SEQ ID NO:6, the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence containing one, two, or three amino acid modifications relative to the amino acid sequence of SEQ ID NO:7, and the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence containing one, two, or three amino acid modifications relative to the amino acid sequence of SEQ ID NO:8. In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO:6, the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, and the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the amino acid sequence of the VH CDR1 consists of the amino acid sequence of SEQ ID NO:6, the amino acid sequence of the VH CDR2 consists of the amino acid sequence of SEQ ID NO:7, and the amino acid sequence of the VH CDR3 consists of the amino acid sequence of SEQ ID NO:8.

[0119] In some embodiments, the VL comprises three CDRs, VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the VL comprises the VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence containing one, two, or three amino acid modifications relative to the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of VL CDR2 comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence containing one, two, or three amino acid modifications relative to the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence containing one, two, or three amino acid modifications relative to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of VL CDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence of the VL CDR1 consists of the amino acid sequence of SEQ ID NO:3, the amino acid sequence of the VL CDR2 consists of the amino acid sequence of SEQ ID NO:4, and the amino acid sequence of the VL CDR3 consists of the amino acid sequence of SEQ ID NO:5.

[0120] In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 of SEQ ID NO:2, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 of SEQ ID NO:1. In some embodiments, the amino acid sequence of the VH CDR1 comprises the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that includes one, two or three amino acid modifications to the amino acid sequence of SEQ ID NO:6; the amino acid sequence of the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that includes one, two or three amino acid modifications to the amino acid sequence of SEQ ID NO:7; the amino acid sequence of the VH CDR3 comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence that includes one, two or three amino acid modifications to the amino acid sequence of SEQ ID NO:8; the amino acid sequence of the VL CDR1 comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence that includes one, two or three amino acid modifications to the amino acid sequence of SEQ ID NO:3; the amino acid sequence of the VL CDR2 comprises the amino acid sequence of SEQ ID NO:4 or an amino acid sequence that includes one, two or three amino acid modifications to the amino acid sequence of SEQ ID NO:4; and the amino acid sequence of the VL CDR3 comprises the amino acid sequence of SEQ ID NO:5 or an amino acid sequence that includes one, two or three amino acid modifications to the amino acid sequence of SEQ ID NO:5.

[0121] In some embodiments, the amino acid sequence of the VH CDR1 comprises the amino acid sequence of SEQ ID NO:6, the amino acid sequence of the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, the amino acid sequence of the VH CDR3 comprises the amino acid sequence of SEQ ID NO:8, the amino acid sequence of the VL CDR1 comprises the amino acid sequence of SEQ ID NO:3, the amino acid sequence of the VL CDR2 comprises the amino acid sequence of SEQ ID NO:4, and the amino acid sequence of the VL CDR3 comprises the amino acid sequence of SEQ ID NO:5.

[0122] In some embodiments, the amino acid sequence of the VH CDR1 consists of the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of the VH CDR2 consists of the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of the VH CDR3 consists of the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of the VL CDR1 consists of the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of the VL CDR2 consists of the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of the VL CDR3 consists of the amino acid sequence of SEQ ID NO: 5.

[0123] In some embodiments, the VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the VH consists of a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence of SEQ ID NO: 2.

[0124] In some embodiments, the VL comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the VL consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the VL consists of the amino acid sequence of SEQ ID NO: 1.

[0125] In some embodiments, the VH comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2, and the VL comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:2, and the VL comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the amino acid sequence of the VH consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2, and the amino acid sequence of the VL consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence of SEQ ID NO:2, and the amino acid sequence of the VL consists of the amino acid sequence of SEQ ID NO:1.

[0126] In some embodiments, the hCD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the hCD19 binding domain comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the hCD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the hCD19 binding domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the hCD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the hCD19 binding domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the hCD19 binding domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the hCD19 binding domain comprises the amino acid sequence of SEQ ID NO:14.In some embodiments, the amino acid sequence of the hCD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the hCD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the hCD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the hCD19 binding domain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the amino acid sequence of the hCD19 binding domain consists of the amino acid sequence of SEQ ID NO: 16.

[0127] In some embodiments, the VH comprises a VH CDR1 encoded by the polynucleotide sequence of SEQ ID NO:24, or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:24, a VH CDR2 encoded by the polynucleotide sequence of SEQ ID NO:25, or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:25, and a VH CDR3 encoded by the polynucleotide sequence of SEQ ID NO:26, or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:26. In some embodiments, the VH comprises a VH CDR1 encoded by the polynucleotide sequence of SEQ ID NO:24, a VH CDR2 encoded by the polynucleotide sequence of SEQ ID NO:25, and a VH CDR3 encoded by the polynucleotide sequence of SEQ ID NO:26.

[0128] In some embodiments, the VL comprises a VL CDR1 encoded by the polynucleotide sequence of SEQ ID NO:21, or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications relative to the polynucleotide acid sequence of SEQ ID NO:21, a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO:22, or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications relative to the polynucleotide acid sequence of SEQ ID NO:22, and a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO:23, or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications relative to the polynucleotide acid sequence of SEQ ID NO:23. In some embodiments, the VL comprises a VL CDR1 encoded by the polynucleotide sequence of SEQ ID NO:21, a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO:22, and a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO:23.

[0129] In some embodiments, the VH comprises a VH CDR1 encoded by a polynucleotide sequence of SEQ ID NO:24 or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:24; a VH CDR2 encoded by a polynucleotide sequence of SEQ ID NO:25 or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:25; a VH CDR3 encoded by a polynucleotide sequence of SEQ ID NO:26 or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:26; and a VL CDR4 encoded by a polynucleotide sequence of SEQ ID NO:21 or a polynucleotide sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO:21. and a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO:22 or a polynucleotide sequence containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotide modifications relative to the polynucleotide acid sequence of SEQ ID NO:22; and a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO:23 or a polynucleotide sequence containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotide modifications relative to the polynucleotide acid sequence of SEQ ID NO:23.

[0130] In some embodiments, the VH comprises a VH CDR1 encoded by the polynucleotide sequence of SEQ ID NO:24, a VH CDR2 encoded by the polynucleotide sequence of SEQ ID NO:25, a VH CDR3 encoded by the polynucleotide sequence of SEQ ID NO:26, a VL CDR1 encoded by the polynucleotide sequence of SEQ ID NO:21, a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO:22, and a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO:23.

[0131] In some embodiments, the VH is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 20. In some embodiments, the VH is encoded by the polynucleotide sequence of SEQ ID NO: 20.

[0132] In some embodiments, the VL is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 19. In some embodiments, the VL is encoded by the polynucleotide sequence of SEQ ID NO:19.

[0133] In some embodiments, the VH is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 20, and the VL is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 19. In some embodiments, the VH is encoded by the polynucleotide sequence of SEQ ID NO: 20, and the VL is encoded by the polynucleotide sequence of SEQ ID NO: 19.

[0134] In some embodiments, the hCD19 binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 29. In some embodiments, the hCD19 binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 30. In some embodiments, the hCD19 binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 31. In some embodiments, the hCD19 binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 32. In some embodiments, the hCD19 binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 33. In some embodiments, the hCD19 binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 34.

[0135] The amino acid and polynucleotide sequences of exemplary hCD19 binding domains are shown in Table 1 herein.

[0136] [Table 1-1]

[0137] [Table 1-2]

[0138] [Table 1-3]

[0139] [Table 1-4]

[0140] 5.2.2 Hinge domain In some embodiments, the CAR comprises an amino acid sequence located between the antigen binding domain and the transmembrane domain, referred to herein as the hinge domain. The hinge domain can provide optimal distance of the antigen binding domain from the membrane of the cell when the CAR is expressed on the cell surface. The hinge domain can also provide optimal flexibility for the antigen binding domain to bind to its target antigen. In some embodiments, the hinge domain is derived from the extracellular region of a naturally occurring protein expressed on the surface of an immune effector cell. In some embodiments, the hinge domain is derived from the hinge domain of a naturally occurring protein expressed on the surface of an immune effector cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell.

[0141] In some embodiments, the hinge domain is operably linked directly to the C-terminus of the antigen binding domain. In some embodiments, the hinge domain is operably linked indirectly to the C-terminus of the antigen binding domain. In some embodiments, the hinge domain is operably linked indirectly to the C-terminus of the antigen binding domain via a peptide linker. In some embodiments, the hinge domain is operably linked directly to the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is operably linked indirectly to the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is operably linked indirectly to the N-terminus of the transmembrane domain via a peptide linker.

[0142] In some embodiments, the hinge domain is derived from human CD8α (hCD8α). In some embodiments, the hinge domain comprises the hinge domain of hCD8α. In some embodiments, the hinge domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the amino acid sequence of the hinge domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the amino acid sequence of the hinge domain consists of the amino acid sequence of SEQ ID NO: 37.

[0143] In some embodiments, the hinge domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the amino acid sequence of the hinge domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the amino acid sequence of the hinge domain consists of the amino acid sequence of SEQ ID NO: 38.

[0144] In some embodiments, the hinge domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 40. In some embodiments, the hinge domain is encoded by the polynucleotide sequence of SEQ ID NO: 40.

[0145] In some embodiments, the hinge domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 41. In some embodiments, the hinge domain is encoded by the polynucleotide sequence of SEQ ID NO:41.

[0146] In some embodiments, the hinge domain is derived from human CD28 (hCD28). In some embodiments, the hinge domain comprises the hinge domain of hCD28. In some embodiments, the hinge domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the amino acid sequence of the hinge domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the amino acid sequence of the hinge domain consists of the amino acid sequence of SEQ ID NO:39. In some embodiments, the hinge domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:42. In some embodiments, the hinge domain is encoded by the polynucleotide sequence of SEQ ID NO:42.

[0147] The amino acid and polynucleotide sequences of exemplary hinge domains are shown in Table 2 herein.

[0148] [Table 2]

[0149] 5.2.3 Transmembrane domains The transmembrane domain of the CAR functions to embed the CAR in the plasma membrane of the cell. In some embodiments, the transmembrane domain is operably linked to the C-terminus of the antigen binding domain. In some embodiments, the transmembrane domain is operably linked directly to the C-terminus of the antigen binding domain. In some embodiments, the transmembrane domain is operably linked indirectly to the C-terminus of the antigen binding domain. In some embodiments, the transmembrane domain is operably linked indirectly to the C-terminus of the antigen binding domain via a peptide linker. In some embodiments, the transmembrane domain is operably linked indirectly to the C-terminus of the antigen binding domain via a hinge domain.

[0150] In some embodiments, the transmembrane domain is operably linked to the C-terminus of the hinge domain. In some embodiments, the transmembrane domain is operably linked directly to the C-terminus of the hinge domain. In some embodiments, the transmembrane domain is operably linked indirectly to the C-terminus of the hinge domain. In some embodiments, the transmembrane domain is operably linked indirectly to the C-terminus of the hinge domain via a peptide linker.

[0151] In some embodiments, the transmembrane domain is operably linked to the N-terminus of the cytoplasmic domain. In some embodiments, the transmembrane domain is operably linked directly to the N-terminus of the cytoplasmic domain. In some embodiments, the transmembrane domain is operably linked indirectly to the N-terminus of the cytoplasmic domain. In some embodiments, the transmembrane domain is operably linked indirectly to the N-terminus of the cytoplasmic domain via a peptide linker.

[0152] In some embodiments, the transmembrane domain is derived from the transmembrane domain of a naturally occurring transmembrane protein expressed on the surface of an immune effector cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the transmembrane domain and the hinge domain are derived from the same naturally occurring transmembrane protein expressed on the surface of an immune effector cell.

[0153] In some embodiments, the transmembrane is derived from a transmembrane domain of a protein selected from the group consisting of CD8α, CD28, TCRα, TCRβ, TCRζ, CD3ε, CD45, CD4, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0154] Alternatively, the transmembrane domain can be synthetic (i.e., not derived from a naturally occurring transmembrane protein). In some embodiments, a synthetic transmembrane domain comprises primarily hydrophobic amino acid residues (e.g., leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0155] In some embodiments, the transmembrane domain comprises the transmembrane domain of hCD8α, or a functional fragment or variant thereof. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the amino acid sequence of the transmembrane domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the amino acid sequence of the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 43.

[0156] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the amino acid sequence of the transmembrane domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the amino acid sequence of the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 44.

[0157] In some embodiments, the transmembrane domain comprises the transmembrane domain of hCD28, or a functional fragment or variant thereof. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the amino acid sequence of the transmembrane domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the amino acid sequence of the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 45.

[0158] In some embodiments, the transmembrane domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 49. In some embodiments, the transmembrane domain is encoded by a polynucleotide sequence of SEQ ID NO: 49. In some embodiments, the transmembrane domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 50. In some embodiments, the transmembrane domain is encoded by a polynucleotide sequence of SEQ ID NO: 50. In some embodiments, the transmembrane domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 51. In some embodiments, the transmembrane domain is encoded by the polynucleotide sequence of SEQ ID NO: 51. In some embodiments, the transmembrane domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 52. In some embodiments, the transmembrane domain is encoded by the polynucleotide sequence of SEQ ID NO:52.

[0159] In some embodiments, a CAR comprises a hinge region and a transmembrane domain that, together, comprise an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, a CAR comprises a hinge region and a transmembrane domain that, together, comprise an amino acid sequence of SEQ ID NO: 46. In some embodiments, the amino acid sequences of the hinge region and the transmembrane domain, together, consist of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the amino acid sequences of the hinge region and the transmembrane domain, together, consist of the amino acid sequence of SEQ ID NO: 46.

[0160] In some embodiments, a CAR comprises a hinge region and a transmembrane domain that, together, comprise an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, a CAR comprises a hinge region and a transmembrane domain that, together, comprise an amino acid sequence of SEQ ID NO: 47. In some embodiments, the amino acid sequences of the hinge region and the transmembrane domain, together, consist of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the amino acid sequences of the hinge region and the transmembrane domain, together, consist of the amino acid sequence of SEQ ID NO: 47.

[0161] In some embodiments, a CAR comprises a hinge region and a transmembrane domain that, together, comprise an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, a CAR comprises a hinge region and a transmembrane domain that, together, comprise an amino acid sequence of SEQ ID NO: 48. In some embodiments, the amino acid sequences of the hinge region and the transmembrane domain, together, consist of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the amino acid sequences of the hinge region and the transmembrane domain, together, consist of the amino acid sequence of SEQ ID NO: 48.

[0162] In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 53. In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence of SEQ ID NO: 53. In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 54. In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence of SEQ ID NO: 54. In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 55. In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence of SEQ ID NO: 55. In some embodiments, the hinge region and transmembrane domain, together, are encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 56. In some embodiments, the hinge region and transmembrane domain, together, are encoded by the polynucleotide sequence of SEQ ID NO: 56.

[0163] Exemplary transmembrane domains and hinges, as well as amino acid and polynucleotide sequences for the transmembrane domains, are shown in Table 3 herein.

[0164] [Table 3]

[0165] 5.2.4 Cytoplasmic domain The cytoplasmic domain of the CAR described herein comprises at least a primary signaling domain that initiates antigen-dependent primary activation, and optionally one or more costimulatory domains to provide a costimulatory signal.

[0166] In some embodiments, the cytoplasmic domain is operably linked to the C-terminus of the transmembrane domain. In some embodiments, the cytoplasmic domain is operably linked directly to the C-terminus of the transmembrane domain. In some embodiments, the cytoplasmic domain is operably linked indirectly to the C-terminus of the transmembrane domain. In some embodiments, the cytoplasmic domain is operably linked indirectly to the C-terminus of the transmembrane domain via a peptide linker.

[0167] In some embodiments, the primary signaling domain comprises at least one immunoreceptor tyrosine-based activation motif (ITAM). Exemplary primary signaling domains include, but are not limited to, the signaling domains of CD3zeta, CD3gamma, CD3delta, CD3epsilon, FcRgamma, FcRbeta, CDS, CD22, CD79a, CD79b, and CD66d, as well as functional fragments and variants thereof. In some embodiments, the primary signaling domain is derived from CD3zeta, CD3gamma, CD3delta, CD3epsilon, FcRgamma, FcRbeta, CDS, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary signaling domain comprises the CD3zeta intracellular signaling domain, or a functional fragment or variant thereof. In some embodiments, the primary signaling domain is derived from human CD3zeta.

[0168] In some embodiments, the cytoplasmic domain comprising the primary signaling domain comprises an amino acid sequence that is at least at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the cytoplasmic domain comprising the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the amino acid sequence of the cytoplasmic domain comprising the primary signaling domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the amino acid sequence of the cytoplasmic domain comprising the primary signaling domain consists of the amino acid sequence of SEQ ID NO: 60.

[0169] In some embodiments, the cytoplasmic domain comprising the primary signaling domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 67. In some embodiments, the cytoplasmic domain comprising the primary signaling domain is encoded by a polynucleotide sequence of SEQ ID NO: 67. In some embodiments, the cytoplasmic domain comprising the primary signaling domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 68. In some embodiments, the cytoplasmic domain comprising the primary signaling domain is encoded by the polynucleotide sequence of SEQ ID NO: 68.

[0170] In some embodiments, the cytoplasmic domain comprises at least one costimulatory domain. In some embodiments, the cytoplasmic domain comprises multiple costimulatory domains. In some embodiments, the cytoplasmic domain comprises a primary signaling domain and one costimulatory domain. In some embodiments, the cytoplasmic domain comprises a primary signaling domain and two costimulatory domains, where the two costimulatory domains may be the same or different. In some embodiments, the cytoplasmic domain comprises a primary signaling domain and three costimulatory domains, where each of the three costimulatory domains may independently be the same or different from another one of the three costimulatory domains.

[0171] In some embodiments, the cytoplasmic domain comprises a costimulatory domain of a protein selected from the group consisting of CD28, 4-IBB, OX40, CD27, CD30, CD40, PD-I, ICOS, LFA1, CD2, CD7, LIGHT, NKG2C, B7-H3, DAP10, and DAPI2, or a functional fragment or variant thereof. In some embodiments, the protein is CD28. In some embodiments, the protein is 4-1BB.

[0172] In some embodiments, the cytoplasmic domain comprises a costimulatory domain of CD28, or a functional fragment or variant thereof. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:57. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:58. In some embodiments, the cytoplasmic domain comprises an amino acid sequence of SEQ ID NO:58. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:57. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of the amino acid sequence of SEQ ID NO:57. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of the amino acid sequence of SEQ ID NO:58.

[0173] In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 64. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence of SEQ ID NO: 64. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 65. In some embodiments, the cytoplasmic domain is encoded by the polynucleotide sequence of SEQ ID NO: 65.

[0174] In some embodiments, the cytoplasmic domain comprises a costimulatory domain of 4-1BB, or a functional fragment or variant thereof. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:59. In some embodiments, the cytoplasmic domain comprises an amino acid sequence of SEQ ID NO:59. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:59. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of the amino acid sequence of SEQ ID NO:59.

[0175] In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 66. In some embodiments, the cytoplasmic domain is encoded by the polynucleotide sequence of SEQ ID NO:66.

[0176] The primary signaling domain may be operably linked directly or indirectly to one or more costimulatory domains. In some embodiments, the primary signaling domain is operably linked directly to the costimulatory domain. In some embodiments, the primary signaling domain is operably linked indirectly to the costimulatory domain. In some embodiments, the primary signaling domain is operably linked indirectly to the costimulatory domain via a peptide linker. In some embodiments, the costimulatory domain is operably linked to the N-terminus of the primary signaling domain. In some embodiments, the costimulatory domain is operably linked directly to the N-terminus of the primary signaling domain. In some embodiments, the costimulatory domain is operably linked indirectly ... via a peptide linker.

[0177] The primary signaling domain may be operably linked directly or indirectly to the transmembrane domain. In some embodiments, the primary signaling domain is operably linked directly to the transmembrane domain. In some embodiments, the primary signaling domain is operably linked indirectly to the transmembrane domain. In some embodiments, the primary signaling domain is operably linked indirectly to the transmembrane domain via a peptide linker.

[0178] The costimulatory domain may be operably linked directly or indirectly to the transmembrane domain. In some embodiments, the costimulatory domain is operably linked directly to the transmembrane domain. In some embodiments, the costimulatory domain is operably linked indirectly to the transmembrane domain. In some embodiments, the costimulatory domain is operably linked indirectly to the transmembrane domain via a peptide linker.

[0179] In some embodiments, the intracellular signaling domain comprises a costimulatory domain of CD28, or a functional variant or fragment thereof, and a signaling domain of CD3zeta, or a functional fragment or variant thereof. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:63. In some embodiments, the cytoplasmic domain comprises the amino acid sequence of SEQ ID NO:63.

[0180] In some embodiments, the amino acid sequence of the cytoplasmic domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of the amino acid sequence of SEQ ID NO: 61. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of the amino acid sequence of SEQ ID NO: 63.

[0181] In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 69. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence of SEQ ID NO: 69. In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 71. In some embodiments, the cytoplasmic domain is encoded by the polynucleotide sequence of SEQ ID NO: 71.

[0182] In some embodiments, the intracellular signaling domain comprises a costimulatory domain of 4-1BB, or a functional variant or fragment thereof, and a primary signaling domain of CD3ζ, or a functional fragment or variant thereof. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:62. In some embodiments, the cytoplasmic domain comprises an amino acid sequence of SEQ ID NO:62. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:62. In some embodiments, the amino acid sequence of the cytoplasmic domain consists of the amino acid sequence of SEQ ID NO:62.

[0183] In some embodiments, the cytoplasmic domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 70. In some embodiments, the cytoplasmic domain is encoded by the polynucleotide sequence of SEQ ID NO: 70.

[0184] The amino acid and polynucleotide sequences of exemplary cytoplasmic domains, including primary signaling domains, costimulatory domains, and intracellular signaling domains, are provided in Table 4 herein.

[0185] [Table 4-1]

[0186] [Table 4-2]

[0187] [Table 4-3]

[0188] 5.2.5 Exemplary CD19-Specific CARs Exemplary CD19-specific CAR amino acid and polynucleotide sequences are provided herein in Table 5. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the CAR comprises an amino acid sequence 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the CAR comprises an amino acid sequence 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the CAR comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CAR comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the CAR comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 81.

[0189] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 81.

[0190] In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the amino acid sequence of the CAR consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:81.

[0191] In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 73. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 74. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 75. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 76. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 79. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 80. In some embodiments, the amino acid sequence of the CAR consists of the amino acid sequence of SEQ ID NO: 81.

[0192] In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 82, 83, 84, 86, 87, 88, 90, 91, 92, 93, 94, or 95. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 82. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 83. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 84. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:86. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 87. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:88.In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 89. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 90. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:91. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 92. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 93. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:94. In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:95.

[0193] In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 82, 83, 84, 86, 87, 88, 90, 91, 92, 93, 94, or 95. In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 82. In some embodiments, the CAR is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 83. In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 84. In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 86. In some embodiments, the CAR is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 87. In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 88. In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 90. In some embodiments, the CAR is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 91. In some embodiments, the CAR is encoded by a polynucleotide sequence of SEQ ID NO: 92. In some embodiments, the CAR is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 93. In some embodiments, the CAR is encoded by the polynucleotide sequence of SEQ ID NO: 94. In some embodiments, the CAR is encoded by the polynucleotide sequence of SEQ ID NO: 95.

[0194] In some embodiments, the CAR comprises the amino acid sequence of the CAR CTL019. In some embodiments, the CAR is the CAR CTL019. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T cell tisagenlecleucel. In some embodiments, the CAR is the CAR expressed by the CAR T cell tisagenlecleucel. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T cell KYMRIAH®. In some embodiments, the CAR is the CAR expressed by the CAR T cell KYMRIAH®. In some embodiments, the CAR comprises the amino acid sequence of the CAR KTE-C19. In some embodiments, the CAR is the CAR KTE-C19. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T cell axicabtagene ciloleucel. In some embodiments, the CAR is the CAR expressed by the CAR T cell axicabtagene ciloleucel. In some embodiments, the CAR comprises the amino acid sequence of a CAR expressed by the CAR T cell YESCARTA®. In some embodiments, the CAR is a CAR expressed by the CAR T cell YESCARTA®.

[0195] Additional exemplary CD19-specific CARs are described, for example, in US89006682, WO2019 / 213282, US2020 / 0268860, WO2020 / 227177, US10457730, WO2019 / 159193, US10287350, US10221245, US2019 / 0125799, WO2018 / 201 794, US2017 / 0368098, US2016 / 0145337, US9701758, WO2014 / 153270, WO2012 / 079000, WO2 019 / 160956, WO2019 / 161796, WO2020 / 222176, WO2020 / 219848, US2019 / 0135894, US10774 388, WO2020 / 180882, US10765701, WO2020 / 172641, WO2020 / 172440, WO2016 / 149578, WO20 20 / 124021, WO2020 / 108646, WO2020 / 108643, WO2020 / 113188, WO2020 / 108644, WO2020 / 10 8645, WO2020 / 108642, US10669549, WO2020 / 102770, US10501539, WO2020 / 069409, US10603380, US10533055, WO2020 / 010235, WO2019 / 246546, the entire contents of each of which are incorporated herein by reference.

[0196] [Table 5-1]

[0197] [Table 5-2]

[0198] [Table 5-3]

[0199] [Table 5-4]

[0200] [Table 5-5]

[0201] [Table 5-6]

[0202] [Table 5-7]

[0203] [Table 5-8]

[0204] [Table 5-9]

[0205] [Table 5-10]

[0206] [Table 5-11]

[0207] [Table 5-12]

[0208] 5.3 Cytokines The present disclosure also provides a recombinant vector comprising a cytokine. In some embodiments, the cytokine is an interleukin. Exemplary interleukins include, but are not limited to, IL-15, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and functional variants and functional fragments thereof. In some embodiments, the cytokine is soluble. In some embodiments, the cytokine is membrane bound.

[0209] In some embodiments, the cytokine is a fusion protein comprising a soluble cytokine, or a functional fragment or variant thereof, operably linked to a soluble form of the cytokine's cognate receptor, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises human IL-15 (hIL-15) operably linked to a soluble form of the human IL-15Rα receptor (hIL-15Rα). This fusion protein is also referred to herein as IL-15 superagonist (IL-15SA). In some embodiments, hIL-15 is operably linked directly to hIL-15Rα. In some embodiments, hIL-15 is operably linked indirectly to a soluble form of hIL-15Rα. In some embodiments, hIL-15 is operably linked indirectly to a soluble form of hIL-15Rα via a peptide linker. In some embodiments, the fusion protein is ALT-803, an IL-15 / IL-15Ra Fc fusion protein. ALT-803 is disclosed in WO2008 / 143794, the entire contents of which are incorporated herein by reference.

[0210] In some embodiments, the cytokine is a fusion protein comprising a soluble cytokine, or a functional fragment or variant thereof, operably linked to a membrane-bound form of the cytokine's cognate receptor, or a functional fragment or variant thereof. In some embodiments, the fusion protein comprises human IL-15 (hIL-15) operably linked to the human IL-15Rα receptor (hIL-15Rα). This fusion protein is also referred to herein as membrane-bound IL-15 (mbIL15). In some embodiments, the hIL-15 is operably linked directly to the hIL-15Rα. In some embodiments, the hIL-15 is operably linked indirectly to the hIL-15Rα. In some embodiments, the hIL-15 is operably linked indirectly to the hIL-15Rα via a peptide linker.

[0211] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 125, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO: 125. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the amino acids of the linker consist of the amino acid sequence of SEQ ID NO: 125, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO: 125. In some embodiments, the amino acids of the linker consist of the amino acid sequence of SEQ ID NO: 125.

[0212] In some embodiments, the linker is encoded by a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 136. In some embodiments, the linker is encoded by the polynucleotide sequence of SEQ ID NO:136.

[0213] In some embodiments, hIL-15 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 123. In some embodiments, hIL-15 comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments, the amino acid sequence of hIL-15 consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the amino acid sequence of hIL-15 consists of the amino acid sequence of SEQ ID NO: 123.

[0214] In some embodiments, IL-15 is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 134. In some embodiments, IL-15 is encoded by the polynucleotide sequence of SEQ ID NO: 134.

[0215] In some embodiments, hIL-15Rα comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, hIL-15Rα comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the amino acid sequence of hIL-15Rα consists of a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the amino acid sequence of hIL-15Rα consists of the amino acid sequence of SEQ ID NO: 124.

[0216] In some embodiments, hIL-15Rα is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 135. In some embodiments, hIL-15Rα is encoded by a polynucleotide sequence of SEQ ID NO: 135. In some embodiments, hIL-15Rα is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 163. In some embodiments, hIL-15Rα is encoded by the polynucleotide sequence of SEQ ID NO: 163.

[0217] In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:119, 120, 121, 122, 180, or 183. In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:119. In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:120. In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:121. In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:122. In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 180. In some embodiments, the fusion protein comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 183. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 180, or 183. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 119. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 120. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 121. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 122. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 180. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 183.

[0218] In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 180, or 183. In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180. In some embodiments, the amino acid sequence of the fusion protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 183. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 180, or 183. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 119. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 121. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 122. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 180. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO: 183.

[0219] In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, or 181. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 126. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 127. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 128. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 129. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 130. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 131. In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 132.In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:181.

[0220] In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, or 181. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 126. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 127. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 128. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 129. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 130. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 131. In some embodiments, the fusion protein is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 132. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 132. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 181.

[0221] Exemplary cytokine fusion proteins and components thereof are disclosed in Table 6. Further exemplary mbIL15 fusions are disclosed in Hurton et al., "Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells," PNAS, 113(48)E7788-E7797 (2016), the entire contents of which are incorporated herein by reference.

[0222] The amino acid and polynucleotide sequences of exemplary cytokine fusion proteins and component polypeptides are provided in Table 6 herein.

[0223] [Table 6-1]

[0224] [Table 6-2]

[0225] [Table 6-3]

[0226] [Table 6-4]

[0227] [Table 6-5]

[0228] [Table 6-6]

[0229] [Table 6-7]

[0230] [Table 6-8]

[0231] [Table 6-9]

[0232] 5.4 Marker proteins The marker proteins described herein function to allow for selective depletion of anti-CD19 CAR-expressing cells in vivo through administration of an agent (e.g., an antibody) that specifically binds to the marker protein and mediates or catalyzes the killing of anti-CD19 CAR-expressing cells. In some embodiments, the marker protein is expressed on the surface of cells expressing the anti-CD19 CAR.

[0233] In some embodiments, the marker protein comprises the extracellular domain of a cell surface protein, or a functional fragment or variant thereof. In some embodiments, the cell surface protein is human epidermal growth factor receptor 1 (hHER1). In some embodiments, the marker protein comprises a truncated HER1 protein that can be bound by an anti-hHER1 antibody. In some embodiments, the marker protein comprises a variant of a truncated hHER1 protein that can be bound by an anti-hHER1 antibody. In some embodiments, the hHER1 marker protein provides a safety mechanism by allowing depletion of infused CAR-T cells by administering an antibody that recognizes the hHER1 marker protein expressed on the surface of anti-CD19 CAR-expressing cells. An exemplary antibody that binds to the hHER1 marker protein is cetuximab.

[0234] In some embodiments, the hHER1 marker protein comprises, from N-terminus to C-terminus, domain III of hHER1, or a functional fragment or functional variant thereof, the N-terminal portion of domain IV of hHER1, and the transmembrane region of human CD28.

[0235] In some embodiments, domain III of hHER1 comprises the amino acid sequence of SEQ ID NO: 98, or the amino acid sequence of SEQ ID NO: 98 with one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of domain III of hHER1 consists of the amino acid sequence of SEQ ID NO: 98, or the amino acid sequence of SEQ ID NO: 98 with one, two, or three amino acid modifications.

[0236] In some embodiments, domain III of hHER1 is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 110. In some embodiments, domain III of hHER1 is encoded by the polynucleotide sequence of SEQ ID NO: 110. In some embodiments, domain III of hHER1 is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 164. In some embodiments, domain III of hHER1 is encoded by the polynucleotide sequence of SEQ ID NO: 164.

[0237] In some embodiments, the N-terminal portion of Domain IV of hHER1 comprises amino acids 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, or 1-10 of SEQ ID NO: 99. In some embodiments, the C-terminus of Domain III of hHER1 is fused directly to the N-terminus of the N-terminal portion of Domain IV of hHER1.

[0238] In some embodiments, the C-terminus of the N-terminal portion of domain IV of hHER1 is indirectly fused to the N-terminus of the CD28 transmembrane domain via a peptide linker. In some embodiments, the peptide linker comprises glycine and serine amino acid residues. In some embodiments, the peptide linker is about 5-25, 5-20, 5-15, 5-10, 10-20, or 10-15 amino acids in length.

[0239] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence that contains one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 102. In some embodiments, the peptide linker is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 114. In some embodiments, the peptide linker is encoded by the polynucleotide sequence of SEQ ID NO: 114.

[0240] In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 97, 103, 104, 166, or 167. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 166. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 167.

[0241] In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 96, 97, 103, or 104. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 166. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 167.

[0242] In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 97, 103, 104, 166, or 167. In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 166. In some embodiments, the marker protein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167.

[0243] In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 96, 97, 103, 104, 166, or 167. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 96. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 103. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 104. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 166. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 167.

[0244] In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 107, 162, 108, 109, 115, 116, 173, or 174. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 107. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 162. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 108. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 109. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:115. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 116. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO:173.In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:174.

[0245] In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 107, 162, 108, 109, 115, 116, 173, or 174. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 107. In some embodiments, the marker protein is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 162. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 108. In some embodiments, the marker protein is encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 109. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 115. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 116. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 173. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO: 174.

[0246] In some embodiments, the marker protein is derived from human CD20 (hCD20). In some embodiments, the marker protein comprises a truncated hCD20 protein comprising the extracellular domain (hCD20t), or a functional fragment or variant thereof. In some embodiments, the hCD20 marker protein provides a safety mechanism by allowing depletion of infused CAR-T cells by administering an antibody that recognizes the hCD20 marker protein expressed on the surface of CAR-expressing cells. An exemplary antibody that binds to the hCD20 marker protein is rituximab.

[0247] In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the marker protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 106.

[0248] In some embodiments, the amino acid sequence of the marker protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the amino acid sequence of the marker protein consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the amino acid sequence of the marker protein consists of the amino acid sequence of SEQ ID NO: 105. In some embodiments, the amino acid sequence of the marker protein consists of the amino acid sequence of SEQ ID NO: 106.

[0249] In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 117 or 118. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 117. In some embodiments, the marker protein is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 118. In some embodiments, the marker protein is encoded by a polynucleotide sequence of SEQ ID NO: 117 or 118. In some embodiments, the marker protein is encoded by a polynucleotide sequence of SEQ ID NO: 117. In some embodiments, the marker protein is encoded by the polynucleotide sequence of SEQ ID NO:118.

[0250] Amino acid and polynucleotide sequences of exemplary marker proteins are provided in Table 7 herein.

[0251] [Table 7-1]

[0252] [Table 7-2]

[0253] [Table 7-3]

[0254] [Table 7-4]

[0255] [Table 7-5]

[0256] [Table 7-6]

[0257] [Table 7-7]

[0258] [Table 7-8]

[0259] [Table 7-9]

[0260] [Table 7-10]

[0261] 5.5 Vectors In one aspect, provided herein is a recombinant vector comprising a polycistronic expression cassette comprising at least three cistrons. In some embodiments, the polycistronic expression cassette comprises at least four, five, or six cistrons. In some embodiments, the polycistronic expression cassette comprises three cistrons. In some embodiments, the polycistronic expression cassette comprises four cistrons. In some embodiments, the polycistronic expression cassette comprises five cistrons.

[0262] In some embodiments, the vector is a non-viral vector.Exemplary non-viral vectors include, but are not limited to, plasmid DNA, episomal plasmid, minicircle, ministring, oligonucleotide (e.g., mRNA, naked DNA).In some embodiments, the polycistronic vector is a DNA plasmid vector.

[0263] In some embodiments, the vector is a viral vector. The viral vector may be replication-competent or replication-incompetent. The viral vector may be integrating or non-integrating. Several viral-based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector may be selected by one skilled in the art. Exemplary viral vectors include, but are not limited to, adenoviral vectors (e.g., adenovirus 5), adeno-associated viral (AAV) vectors (e.g., AAV2, 3, 5, 6, 8, 9), retroviral vectors (MMSV, MSCV), lentiviral vectors (e.g., HIV-1, HIV-2), gamma retroviral vectors, herpes virus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vectors (e.g., MV-Edm), Newcastle disease virus vectors, poxvirus vectors (e.g., VV), measles virus, and picornavirus vectors (e.g., coxsackievirus).

[0264] In one aspect, the vector comprises a polycistronic expression cassette comprising, from 5' to 3', a first polynucleotide sequence encoding a chimeric antigen receptor (CAR), a second polynucleotide sequence comprising an F2A element, a third polynucleotide sequence encoding a cytokine, a fourth polynucleotide sequence comprising a T2A element, and a fifth polynucleotide sequence encoding a marker protein.

[0265] In some embodiments, the F2A element comprises a polynucleotide sequence that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the F2A element comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 137. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 141. In some embodiments, the F2A element comprises the polynucleotide sequence of SEQ ID NO: 141.

[0266] In some embodiments, the F2A element comprises a polynucleotide sequence that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the F2A element comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 138. In some embodiments, the F2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 142. In some embodiments, the F2A element comprises the polynucleotide sequence of SEQ ID NO: 142.

[0267] In some embodiments, the T2A element comprises a polynucleotide sequence that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the T2A element comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 139. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 143. In some embodiments, the T2A element comprises the polynucleotide sequence of SEQ ID NO: 143.

[0268] In some embodiments, the T2A element comprises a polynucleotide sequence encoding an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 140 or 182. In some embodiments, the T2A element comprises a polynucleotide sequence encoding an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the T2A element comprises a polynucleotide sequence encoding an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 182. In some embodiments, the T2A element comprises the amino acid sequence of the polynucleotide sequence SEQ ID NO: 140 or 182. In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140. In some embodiments, the T2A element comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 182.

[0269] In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 144, 145, or 165. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 144. In some embodiments, the T2A element comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 145. In some embodiments, the T2A element comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 165. In some embodiments, the T2A element comprises a polynucleotide sequence of SEQ ID NO: 144, 145, or 165. In some embodiments, the T2A element comprises a polynucleotide sequence of SEQ ID NO: 144. In some embodiments, the T2A element comprises a polynucleotide sequence of SEQ ID NO: 145. In some embodiments, the T2A element comprises a polynucleotide sequence of SEQ ID NO: 165.

[0270] Exemplary polynucleotide sequences encoding the F2A and P2A elements are provided in Table 8 herein.

[0271] [Table 8]

[0272] In some embodiments, the vector or polycistronic expression cassette comprises one or more additional elements, including but not limited to, promoters, enhancers, polyadenylation (polyA) sequences, and selection genes.

[0273] In some embodiments, the vector comprises a polynucleotide sequence encoding a selectable marker that confers a specific trait to cells in which the selectable marker is expressed, allowing for the artificial selection of those cells. Exemplary selectable markers include, but are not limited to, antibiotic resistance (e.g., resistance to kanamycin, ampicillin, or triclosan) genes.

[0274] In some embodiments, the polycistronic expression cassette comprises a transcriptional regulatory element. Exemplary transcriptional regulatory elements include, but are not limited to, a promoter and an enhancer. In some embodiments, the polycistronic expression cassette comprises a promoter sequence 5' of the first 5' cistron. In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 146. In some embodiments, the promoter comprises a polynucleotide sequence of SEQ ID NO: 146. In some embodiments, the promoter polynucleotide sequence consists of a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 146. In some embodiments, the promoter polynucleotide sequence consists of the polynucleotide sequence of SEQ ID NO: 146.

[0275] In some embodiments, the polycistronic expression cassette comprises a polyA sequence 3' of the 3'-terminal cistron. In some embodiments, the polyA sequence comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 148. In some embodiments, the polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 148. In some embodiments, the polyA sequence consists of a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 148. In some embodiments, the polyA sequence consists of the nucleic acid sequence of SEQ ID NO: 148.

[0276] Polynucleotide sequences of exemplary promoters and polyA sequences are provided in Table 9 herein.

[0277] [Table 9]

[0278] Exemplary polycistronic expression cassette polynucleotide sequences are provided herein in Table 10. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 149, 150, or 151. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of Expression No. 149. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of Expression No. 150. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of Expression No. 151.

[0279] In some embodiments, the polycistronic expression cassette comprises the polynucleotide sequence of SEQ ID NO: 149, 150, or 151. In some embodiments, the polycistronic expression cassette comprises the polynucleotide sequence of SEQ ID NO: 149. In some embodiments, the polycistronic expression cassette comprises the polynucleotide sequence of SEQ ID NO: 150. In some embodiments, the polycistronic expression cassette comprises the polynucleotide sequence of SEQ ID NO: 151.

[0280] [Table 10-1]

[0281] [Table 10-2]

[0282] [Table 10-3]

[0283] Amino acid sequences encoded by polynucleotide sequences of exemplary polycistronic expression cassettes are provided herein in Table 11. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 152, 153, or 154. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 152. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 153. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence encoding an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:154.

[0284] In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 152, 153, or 154. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 152. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 153. In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 154.

[0285] [Table 11-1]

[0286] [Table 11-2]

[0287] 5.6 Transposon and transposase systems In some embodiments, the transgenes of the polycistronic vectors are introduced into immune effector cells via synthetic DNA transposable elements, such as DNA transposon / transposase systems, such as Sleeping Beauty (SB). SB belongs to the Tc1 / Mariner superfamily of DNA transposons. DNA transposons translocate from one DNA site to another in a simple cut-and-paste manner. Transposition is the precise process by which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or a different DNA molecule or genome.

[0288] Exemplary DNA transposon / transposase systems include, but are not limited to, Sleeping Beauty (see, e.g., US6489458, US8227432, the contents of each of which are incorporated herein by reference in their entireties), the piggy Bac transposon system (see, e.g., US9228180, Wilson et al, "PiggyBac Transposon-mediated Gene Transfer in Human Cells," Molecular Therapy, 15:139-145 (2007) the contents of each of which are incorporated herein by reference in their entireties), the piggyBat transposon system (see, e.g., Mitra et al., "Functional characterization of piggy Bat from the bat Myotis lucifugus unveils an active mammalian DNA transposon," Proc. Natl. Acad. Sci USA, 1999, 14:111-112 (2007) the contents of which are incorporated herein by reference in their entireties), and the piggyBat transposon system (see, e.g., Mitra et al., "Functional characterization of piggy Bat from the bat Myotis lucifugus unveils an active mammalian DNA transposon," Proc. Natl. Acad. Sci USA, 1999, 14:111-112 (2007) the contents of which are incorporated herein by reference in their entireties). 110:234-239 (2013)), TcBuster (see, e.g., Woodard et al. "Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells," PLOS ONE, 7(11):e42666 (Nov. 2012), the contents of each of which are incorporated herein by reference in their entireties), and the Tol2 transposon system (see, e.g., Kawakami, "Tol2: a versatile gene transfer vector in vertebrates," Genome Biol. 2007;8(Suppl 1):S7, the contents of each of which are incorporated herein by reference in their entireties).Further exemplary transposon / transposase systems are provided in US7148203, US8227432, US20110117072, Mates et al., Nat Genet, 41(6):753-61 (2009), and Ivies et al., Cell, 91(4):501-10, (1997), the contents of each of which are incorporated herein by reference in their entireties.

[0289] In some embodiments, the transgenes described herein are introduced into immune effector cells via the SB transposon / transposase system. The SB transposon system includes SB transposase and SB transposon. The SB transposon system may include naturally occurring SB transposase, or derivatives, variants, and / or fragments that retain activity, and naturally occurring SB transposons, or derivatives, variants, and / or fragments that retain activity. An exemplary SB system is described in Hackett et al., "A Transposon and Transposase System for Human Application," Mol Ther 18:674-83, (2010), the entire contents of which are incorporated herein by reference.

[0290] In some embodiments, the vector comprises a left inverted terminal repeat (ITR) (i.e., the ITR that is 5' to the expression cassette) and a right ITR (i.e., the ITR that is 3' to the expression cassette). The left and right ITR flank the polycistronic expression cassette of the vector. In some embodiments, the left ITR is in the opposite orientation to the polycistronic expression cassette and the right ITR is in the same orientation to the polycistronic expression cassette. In some embodiments, the right ITR is in the opposite orientation to the polycistronic expression cassette and the left ITR is in the same orientation to the polycistronic expression cassette.

[0291] In some embodiments, the left and right ITRs are ITRs of a DNA transposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, TcBuster transposon, and Tol2 transposon. In some embodiments, the left and right ITRs are ITRs of a Sleeping Beauty DNA transposon.

[0292] In some embodiments, the left ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 155 or 156. In some embodiments, the left ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 155. In some embodiments, the left ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 156. In some embodiments, the left ITR comprises the polynucleotide sequence of SEQ ID NO: 156. In some embodiments, the right ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 157, 159, or 184. In some embodiments, the right ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 157. In some embodiments, the right ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 159. In some embodiments, the right ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 184. In some embodiments, the right ITR comprises the polynucleotide sequence of SEQ ID NO: 157. In some embodiments, the right ITR comprises the polynucleotide sequence of SEQ ID NO: 159. In some embodiments, the right ITR comprises the polynucleotide sequence of SEQ ID NO: 184.

[0293] Exemplary polynucleotide sequences of SB ITRs are provided in Table 12 herein.

[0294] [Table 12]

[0295] In some embodiments, the DNA transposase is an SB transposase. In some embodiments, the SB transposase is selected from the group consisting of SB11, SB100X, hSB110, and hSB81. In some embodiments, the SB transposase is SB11. Exemplary SB transposases are described in US9840696, US2016 / 0264949, US9228180, WO2019 / 038197, US10174309, and US10570382, the entire contents of each of which are incorporated herein by reference.

[0296] In some embodiments, the DNA transposase comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the DNA transposase comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the amino acid sequence of the DNA transposase consists of a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the amino acid sequence of the DNA transposase consists of the amino acid sequence of SEQ ID NO: 160.

[0297] In some embodiments, the DNA transposase comprises an amino acid sequence lacking its N-terminal methionine. In some embodiments, the DNA transposase comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 160, lacking its N-terminal methionine, i.e., amino acids 2-340 of SEQ ID NO: 160. In some embodiments, the DNA transposase comprises an amino acid sequence of SEQ ID NO: 160, lacking its N-terminal methionine, i.e., amino acids 2-340 of SEQ ID NO: 160. In some embodiments, the amino acid sequence of the DNA transposase consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 160, lacking its N-terminal methionine, i.e., amino acids 2-340 of SEQ ID NO: 160. In some embodiments, the amino acid sequence of the DNA transposase consists of the amino acid sequence of SEQ ID NO:160, lacking its N-terminal methionine, i.e., amino acids 2 to 340 of SEQ ID NO:160.

[0298] In some embodiments, the DNA transposase is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 161. In some embodiments, the DNA transposase is encoded by the polynucleotide sequence of SEQ ID NO:161.

[0299] In some embodiments, the DNA transposase is encoded by a polynucleotide that is introduced into the cell. In some embodiments, the polynucleotide that encodes the DNA transposase is a DNA vector. In some embodiments, the polynucleotide that encodes the DNA transposase is an RNA vector. In some embodiments, the DNA transposase is encoded on a first vector and the transgene is encoded on a second vector. In some embodiments, the DNA transposase is directly introduced into the cell population as a polypeptide.

[0300] Exemplary SB transposase amino acid and polynucleotide sequences are provided in Table 13 herein.

[0301] [Table 13]

[0302] 5.7 Immune Effector Cells and Methods of Manipulation In one aspect, provided herein is a cell, e.g., an immune effector cell, comprising a recombinant vector (e.g., a vector described herein) comprising a polycistronic expression cassette. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a CD4+ T cell. In some embodiments, the immune effector cell is a CD8+ T cell. In one aspect, provided herein is a population immune effector cell comprising a polycistronic vector described herein. In some embodiments, the population of immune effector cells comprises CD4+ T cells and CD8+ T cells. In some embodiments, the population of immune effector cells is an ex vivo culture.

[0303] In one aspect, provided herein is a method of introducing a vector described herein into a plurality of cells, e.g., immune effector cells, to produce a plurality of engineered cells, e.g., immune effector cells. Methods of introducing and expressing a vector into a cell are well known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., a mammalian (e.g., human), by any method in the art. For example, the expression vector can be transferred into the host cell by transfection or transduction. Exemplary methods for introducing a vector into a host cell include, but are not limited to, electroporation (also referred to herein as electrotransfer), calcium phosphate precipitation, lipofection, particle bombardment, microinjection, mechanical deformation by passing through a microfluidic device, and the like, see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001), the entire contents of which are incorporated herein by reference. In some embodiments, a polycistronic vector is introduced into an immune effector cell, or a population of immune effector cells, via electroporation. Alternative delivery systems include, for example, colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In some embodiments, the polycistronic vector is introduced into a population of cells, e.g., immune effector cells, ex vivo, in vitro, or in vivo. In some embodiments, the polycistronic vector is introduced into a population of cells, e.g., immune effector cells, ex vivo.

[0304] 5.7.1 Sources of immune effector cells Immune effector cells can be obtained from a subject by any suitable method known in the art. For example, T cells (e.g., CD4+ T cells and CD8+ T cells) can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, immune effector cells (e.g., T cells) are obtained from blood collected from a subject using any number of techniques known to those skilled in the art. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a Percoll gradient, or by counterflow centrifugal elutriation.

[0305] Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer (e.g., phosphate-buffered saline (PBS)) or medium for subsequent processing steps. The washing step may be accomplished by methods known to those skilled in the art, such as, for example, by using a semi-automated "flow-through" centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free PBS, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0306] Specific subpopulations of cells may be further isolated by positive or negative selection techniques (e.g., antibody-coated beads, flow cytometry, etc.). In some embodiments, specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques (e.g., antibody-coated beads, flow cytometry, etc.).

[0307] 5.7.2 Activation and Expansion In some embodiments, the T cells are activated prior to introduction of the polycistronic vector described herein. In some embodiments, the T cells are activated by contacting the cells with a molecule that specifically binds to CD3, optionally in combination with a molecule that specifically binds to CD28. An exemplary activation method includes ex vivo contacting the T cells with beads that are covalently linked to anti-CD3 and, optionally, anti-CD28 antibodies. In some embodiments, the T cells are expanded after introduction of the polycistronic vector described herein. In some embodiments, the expansion includes contacting the cells with a molecule that specifically binds to CD3, optionally in combination with a molecule that specifically binds to CD28. An exemplary activation method includes ex vivo contacting the T cells with beads that are covalently linked to anti-CD3 and, optionally, anti-CD28 antibodies.

[0308] 5.7.3 Rapid Personalized Manufacturing (RPM) In one aspect, provided herein is a method of introducing a polycistronic vector described herein into a population of cells to produce an engineered cell population. In some embodiments, the cell population comprises immune effector cells. In some embodiments, the immune effector cells are T cells. In some embodiments, the cell population comprises CD8+ T cells. In some embodiments, the cell population comprises CD4+ T cells. In some embodiments, the cell population comprises CD8+ T cells and CD8+ T cells.

[0309] In some embodiments, the method includes introducing a recombinant vector described herein and a DNA transposase (e.g., a DNA transposase described herein), or a polynucleotide encoding a DNA transposase (e.g., a DNA transposase described herein), into a cell population, and culturing the cell population under conditions in which the transposase integrates the polycistronic expression cassette into the genome of the cell population. In some embodiments, the recombinant vector and the DNA transposase, or a polynucleotide encoding the DNA transposase, are introduced into the cell population using electrotransfer, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, mechanical deformation by passage through a microfluidic device, or a colloidal dispersion system.

[0310] In some embodiments, the engineered cell population is produced in about 1-5 days, 1-4 days, 1-3 days, or 1-2 days. In some embodiments, the engineered cell population is produced in less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments, the engineered cell population is produced in more than 1 day, 2 days, 3 days, 4 days, or 5 days.

[0311] In some embodiments, the cells are not exogenously activated ex vivo. In some embodiments, the cells are not cultured in the presence of exogenous cytokines ex vivo. In some embodiments, the polycistronic vector is introduced (e.g., by electroporation) into resting T cells ex vivo. In some embodiments, the T cells express CCR7 on the cell surface and do not express detectable levels of CD45RO.

[0312] In some embodiments, the cells are cultured ex vivo for 96 hours or less, 72 hours or less, 48 ​​hours or less, 24 hours or less, 12 hours or less, or 6 hours or less after introduction (e.g., by electroporation) of a polycistronic vector described herein. In some embodiments, the cells are cultured ex vivo for about 96 hours, about 72 hours, about 48 hours, about 24 hours, about 12 hours, or about 6 hours after introduction (e.g., by electroporation) of a polycistronic vector described herein. In some embodiments, the cells are cultured ex vivo for about 6-96 hours, about 6-72 hours, about 6-48 hours, about 6-24 hours, about 6-12 hours, about 12-96 hours, about 12-72 hours, about 12-48 hours, about 12-24 hours, about 24-96 hours, about 24-72 hours, about 24-48 hours, about 48-96 hours, or about 48-72 hours after introduction (e.g., by electroporation) of a polycistronic vector described herein.

[0313] In some embodiments, the cells are administered to a subject in need thereof 96 hours or less, 72 hours or less, 48 ​​hours or less, 24 hours or less, 12 hours or less, or 6 hours or less after introduction (e.g., by electroporation) of a polycistronic vector described herein. In some embodiments, the cells are administered to a subject in need thereof about 96 hours, about 72 hours, about 48 hours, about 24 hours, about 12 hours, or about 6 hours after introduction (e.g., by electroporation) of a polycistronic vector described herein. In some embodiments, the cells are administered to a subject in need thereof about 6-96 hours, about 6-72 hours, about 6-48 hours, about 6-24 hours, about 6-12 hours, about 12-96 hours, about 12-72 hours, about 12-48 hours, about 12-24 hours, about 24-96 hours, about 24-72 hours, about 24-48 hours, about 48-96 hours, or about 48-72 hours after introduction (e.g., by electroporation) of a polycistronic vector described herein.

[0314] 5.8 Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising a population of engineered immune effector cells disclosed herein having a desired purity in a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum, albumin, and the like. Examples of suitable surfactants include proteins such as amine, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterion metal complexes such as sodium (e.g., zinc-protein complexes), and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0315] The pharmaceutical compositions described herein may be useful for inducing an immune response in a subject and treating a condition such as cancer. In one embodiment, the present disclosure provides a pharmaceutical composition comprising a population of engineered immune effector cells described herein for use as a medicament. In another embodiment, the present disclosure provides a pharmaceutical composition for use in a method for treating cancer. In some embodiments, the pharmaceutical composition comprises a population of engineered immune effector cells disclosed herein, and optionally one or more additional prophylactic or therapeutic agents in a pharma- ceutically acceptable carrier.

[0316] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include parenteral administration (e.g., intravenous, subcutaneous, intramuscular). In some embodiments, the pharmaceutical composition is formulated for intravenous administration. Injections can be prepared in conventional forms, either liquid solutions or suspensions. Injections may contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0317] In some embodiments, the pharmaceutical composition is formulated for intravenous administration. Suitable carriers for intravenous administration include physiological saline or phosphate buffered saline (PBS), and solutions containing viscosity enhancers and solubilizers, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0318] Formulations to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0319] Pharmaceutically acceptable carriers used in parenteral formulations include, for example, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestrants or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, can be added to parenteral formulations packaged in multi-dose containers. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0320] The exact dose used in the pharmaceutical composition also depends on the route of administration and the severity of the condition caused by it, and should be determined according to the judgment of the practitioner and the circumstances of each subject.For example, the effective dose can also vary depending on the means of administration, the target site, the physiological condition of the subject (including age, weight, and health), other medicines administered, or whether the treatment is preventive or therapeutic.The treatment dosage can be optimally titrated to optimize safety and effectiveness.

[0321] 5.9 Therapeutic Uses and Applications In another aspect, the disclosure provides a method of inducing an immune response in a subject in need thereof comprising administering a population of engineered immune effector cells, vector, polynucleotide, or pharmaceutical composition described herein. In some embodiments, the subject has cancer. In another aspect, the disclosure provides a method of treating a disease or disorder, e.g., cancer or an autoimmune disease or disorder, in a subject in need thereof comprising administering a population of engineered immune effector cells, vector, polynucleotide, or pharmaceutical composition described herein. In another aspect, the disclosure provides a method of treating a disease or disorder, e.g., cancer or an autoimmune disease or disorder, in a subject in need thereof comprising administering a population of engineered immune effector cells, vector, polynucleotide, or pharmaceutical composition described herein.

[0322] In some embodiments, the cells are autologous to the subject receiving said population of engineered immune effector cells, hi some embodiments, the cells are allogeneic to the subject receiving said population of engineered immune effector cells.

[0323] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is associated with expression or overexpression of CD19 on the surface of cancer cells compared to non-cancer cells. In some embodiments, the disease or disorder is a hematological cancer. In some embodiments, the hematological cancer is a leukemia or lymphoma, such as acute leukemia, acute lymphoma, chronic leukemia, or chronic lymphoma. Exemplary cancers include, but are not limited to, cancers associated with expression of CD19, B-cell acute lymphoblastic leukemia (B-ALL) (also known as B-cell acute lymphoblastic leukemia or B-cell acute lymphocytic leukemia), B-lymphoblastic leukemia with t(v;11q23.3), KMT2A-rearranged B-acute lymphoblastic leukemia with t(v;11q23.3), KMT2A-rearranged T-cell acute lymphoblastic leukemia (T-ALL) (also known as T-cell acute lymphoblastic leukemia or T-cell acute lymphocytic leukemia), acute lymphocytic leukemia (ALL) (also known as acute lymphoblastic leukemia or acute lymphocytic leukemia), Ph-like lymphocytic leukemia (Ph-like ALL) (also known as Ph-like acute lymphoblastic leukemia or Ph-like acute lymphocytic leukemia), chronic myeloid leukemia, Myeloma (CML), chronic lymphocytic leukemia (CLL) (also known as chronic lymphoblastic leukemia or chronic lymphocytic leukemia), chronic lymphocytic leukemia, small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal (e.g., thymic) large B-cell lymphoma ( These include: PMBCL), follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and minimal residual disease.

[0324] In some embodiments, the hematological cancer is a B-cell cancer. In some embodiments, the B-cell cancer is a leukemia or lymphoma. In some embodiments, the hematological malignancy is B-ALL, T-ALL, ALL, CLL, SLL, NHL, DLBCL, acute biphenotypic leukemia, or minimal residual disease.

[0325] In some embodiments, the cancer is a recurrent cancer. In some embodiments, the recurrent cancer is associated with expression or overexpression of CD19 on the surface of cancer cells compared to non-cancerous cells. In some embodiments, the disease or disorder is a recurrent hematological cancer. In some embodiments, the recurrent hematological cancer is a recurrent leukemia or a recurrent lymphoma. Exemplary recurrent cancers include, but are not limited to, recurrent cancers associated with expression of CD19, recurrent B-cell acute lymphoblastic leukemia (recurrent B-ALL) (also known as recurrent B-cell acute lymphoblastic leukemia or recurrent B-cell acute lymphoblastic leukemia), recurrent B-lymphoblastic leukemia with t(v;11q23.3), KMT2A-rearranged recurrent B-acute lymphoblastic leukemia with t(v;11q23.3), KMT2A-rearranged recurrent T-cell acute lymphoblastic leukemia (recurrent relapsed acute lymphocytic leukemia (relapsed ALL) (also known as relapsed acute lymphoblastic leukemia or relapsed acute lymphocytic leukemia), relapsed Ph-like lymphocytic leukemia (relapsed Ph-like ALL) (also known as relapsed Ph-like acute lymphoblastic leukemia or relapsed Ph-like acute lymphocytic leukemia), relapsed chronic myeloid leukemia (relapsed CML), relapsed chronic recurrent chronic lymphocytic leukemia (relapsed CLL) (also known as relapsed chronic lymphoblastic leukemia or relapsed chronic lymphocytic leukemia), relapsed chronic lymphocytic leukemia, relapsed small lymphocytic lymphoma (relapsed SLL), relapsed B-cell prolymphocytic leukemia, relapsed blastic plasmacytoid dendritic cell neoplasm, relapsed Burkitt's lymphoma, relapsed diffuse large B-cell lymphoma (relapsed DLBCL), relapsed primary mediastinal (e.g., thymic) large B-cell lymphoma (relapsed PMBCL), These include recurrent follicular lymphoma, recurrent hairy cell leukemia, recurrent small cell follicular lymphoma, recurrent large cell follicular lymphoma, recurrent MALT lymphoma, recurrent mantle cell lymphoma, recurrent marginal zone lymphoma, recurrent multiple myeloma, recurrent myelodysplasia, recurrent myelodysplastic syndrome, recurrent non-Hodgkin's lymphoma (NHL), recurrent plasmablastic lymphoma, recurrent plasmacytoid dendritic cell neoplasm, recurrent Waldenstrom's macroglobulinemia, and recurrent minimal residual disease.

[0326] In some embodiments, the recurrent hematological cancer is a recurrent B-cell cancer. In some embodiments, the recurrent hematological malignancy is recurrent B-ALL, recurrent T-ALL, recurrent ALL, recurrent CLL, recurrent SLL, recurrent NHL, recurrent DLBCL, recurrent acute biphenotypic leukemia, or recurrent minimal residual disease.

[0327] In some embodiments, the cancer is a refractory cancer, e.g., a cancer that is resistant to a treatment (e.g., a standard of care treatment) or that becomes resistant to a treatment over time. In some embodiments, the refractory cancer is associated with expression or overexpression of CD19 on the surface of cancer cells compared to non-cancer cells. In some embodiments, the disease or disorder is a refractory hematological cancer. In some embodiments, the refractory hematological cancer is a refractory leukemia or a refractory lymphoma.Exemplary refractory cancers include, but are not limited to, refractory cancers associated with expression of CD19, refractory B-cell acute lymphoblastic leukemia (refractory B-ALL) (also known as refractory B-cell acute lymphoblastic leukemia or refractory B-cell acute lymphocytic leukemia), refractory B-lymphoblastic leukemia with t(v;11q23.3), KMT2A-rearranged refractory B-acute lymphoblastic leukemia with t(v;11q23.3), KMT2A-rearranged refractory T-cell acute lymphoblastic leukemia (refractory Refractory T-ALL) (also known as refractory T-cell acute lymphoblastic leukemia or refractory T-cell acute lymphocytic leukemia), refractory acute lymphocytic leukemia (refractory ALL) (also known as refractory acute lymphoblastic leukemia or refractory acute lymphocytic leukemia), refractory Ph-like lymphocytic leukemia (refractory Ph-like ALL) (also known as refractory Ph-like acute lymphoblastic leukemia or refractory Ph-like acute lymphocytic leukemia), refractory chronic myeloid leukemia (refractory CML), refractory chronic refractory chronic lymphocytic leukemia (refractory CLL) (also known as refractory chronic lymphoblastic leukemia or refractory chronic lymphocytic leukemia), refractory chronic lymphocytic leukemia, refractory small lymphocytic lymphoma (refractory SLL), refractory B-cell prolymphocytic leukemia, refractory blastic plasmacytoid dendritic cell neoplasm, refractory Burkitt's lymphoma, refractory diffuse large B-cell lymphoma (refractory DLBCL), refractory primary mediastinal (e.g., thymic) large B-cell lymphoma (refractory PMBCL), These include refractory follicular lymphoma, refractory hairy cell leukemia, refractory small cell follicular lymphoma, refractory large cell follicular lymphoma, refractory MALT lymphoma, refractory mantle cell lymphoma, refractory marginal zone lymphoma, refractory multiple myeloma, refractory myelodysplasia, refractory myelodysplastic syndrome, refractory non-Hodgkin's lymphoma (NHL), refractory plasmablastic lymphoma, refractory plasmacytoid dendritic cell neoplasm, refractory Waldenstrom's macroglobulinemia, and refractory minimal residual disease.

[0328] In some embodiments, the refractory hematological cancer is a refractory B-cell cancer. In some embodiments, the refractory hematological malignancy is refractory B-ALL, refractory T-ALL, refractory ALL, refractory CLL, refractory SLL, refractory NHL, refractory DLBCL, refractory acute biphenotypic leukemia, or refractory minimal residual disease.

[0329] In some embodiments, the disease or disorder is an autoimmune disease or disorder, such as a relapsing autoimmune disease or disorder, or a refractory autoimmune disease or disorder.

[0330] In some embodiments, the population of engineered cells is administered to the subject after hematopoietic stem cell transplantation.

[0331] In some embodiments, the population of engineered cells is administered to a subject in combination with (e.g., prior to, concurrently with, or after) one or more additional prophylactic or therapeutic agents. In some embodiments, the therapeutic agent is a chemotherapeutic agent, an anti-cancer agent, an anti-angiogenic agent, an anti-fibrotic agent, an immunotherapeutic agent, a therapeutic antibody, a bispecific antibody, an "antibody-like" therapeutic protein (e.g., DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®, Fab derivatives), an antibody-drug conjugate (ADC), a radiotherapeutic agent, an anti-neoplastic agent, an anti-proliferative agent, an oncolytic virus, a gene modifying or editing agent (e.g., CRISPR / Cas9, zinc finger nucleases or synthetic nucleases, or TALENs), a CAR T cell immunotherapy agent, an engineered T cell receptor (TCR-T), or any combination thereof. In some embodiments, the therapeutic agent is an anti-cancer agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. These therapeutic agents can be in the form of chemical compounds, antibodies, polypeptides, or polynucleotides.

[0332] In some embodiments, the population of engineered immune effector cells, vector, polynucleotide, or pharmaceutical composition is administered to the subject after administration of a lymphodepleting preparation regimen. In some embodiments, the lymphodepleting preparation regimen comprises at least one chemotherapeutic agent. In some embodiments, the lymphodepleting preparation regimen comprises at least two different chemotherapeutic agents. In some embodiments, the lymphodepleting preparation regimen comprises cyclophosphamide. In some embodiments, the lymphodepleting preparation regimen comprises cyclophosphamide administered to the subject in an amount sufficient to reduce an immune response in the subject. In some embodiments, the lymphodepleting preparation regimen comprises fludarabine. In some embodiments, the lymphodepleting preparation regimen comprises fludarabine administered to the subject in an amount sufficient to reduce an immune response in the subject. In some embodiments, the lymphodepleting preparation regimen comprises cyclophosphamide and fludarabine. In some embodiments, the lymphodepleting preparation regimen comprises cyclophosphamide and fludarabine, each administered to the subject in an amount sufficient to reduce an immune response in the subject.

[0333] 5.10 Kits In one aspect, provided herein is a kit comprising one or more of the pharmaceutical compositions, engineered immune effector cell populations, polynucleotides, or vectors described herein and instructions for use. Such kits may include carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, etc. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers may be formed from a variety of materials, such as glass or plastic.

[0334] In specific embodiments, provided herein are pharmaceutical kits comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, the population of engineered immune effector cells, polynucleotides, or vectors provided herein. In one embodiment, the kit comprises a pharmaceutical composition comprising a population of engineered immune effector cells described herein. In one embodiment, the kit comprises a pharmaceutical composition comprising a population of immune effector cells engineered according to the methods described herein. In some embodiments, the kit contains a pharmaceutical composition described herein, a prophylactic or therapeutic agent, and a drug. Optionally, a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products may be associated with such containers, the notice reflecting the agency's approval of manufacture, use, or sale for human administration. EXAMPLES

[0335] The examples in this section (ie, Section 6) are offered by way of illustration and not by way of limitation.

[0336] 6.1 Example 1: Construction of transposon plasmids encoding CD19CAR, mbIL15, and HER1t To improve the uniformity and product manufacturability of multigene co-expression, recombinant nucleic acid Sleeping Beauty transposon plasmids were constructed that contain polycistronic expression cassettes. The polycistronic expression plasmids each contain a transcriptional regulatory element operably linked to a polynucleotide encoding an anti-CD19 CAR (CD19CAR) of SEQ ID NO: 72, a membrane-bound IL-15 / IL-15Rα fusion protein (mbIL15) of SEQ ID NO: 119, and a "kill switch" marker protein (HER1t) of SEQ ID NO: 96 or SEQ ID NO: 166, each separated by an F2A or T2A element that mediates ribosomal skipping to allow expression of separate polypeptide chains. Schematic diagrams of each of the encoded proteins are shown in Figures 1A-1C, from N-terminus (left) to C-terminus (right), respectively.

[0337] Briefly, CD19CAR was generated using the light chain variable region (VL) (SEQ ID NO: 1) and heavy chain variable region (VH) (SEQ ID NO: 2) of mouse monoclonal antibody FMC63. The VL was placed at the mature N-terminus of CD19CAR and connected to the VL via a Whitlow linker peptide (SEQ ID NO: 9), with the human GM-CSF receptor alpha chain signal sequence (SEQ ID NO: 10) at the N-terminus of the VL. The resulting scFv was connected, from N-terminus to C-terminus, to a human CD8α hinge domain (SEQ ID NO: 37), a human CD8α transmembrane domain (SEQ ID NO: 43), a human CD28 cytoplasmic domain (SEQ ID NO: 57), and a human CD3ζ cytoplasmic domain (SEQ ID NO: 60). To enhance CAR expression, the amino acid sequence of the human CD28 cytoplasmic domain was modified to incorporate the amino acid sequence Gly-Gly at amino acids 7-8 of SEQ ID NO: 57, rather than the wild-type sequence Leu-Leu.

[0338] mbIL15 was constructed by connecting human IL-15 (SEQ ID NO: 123) to human IL-15Ra (SEQ ID NO: 124) via a Gly-Ser-rich linker peptide (SEQ ID NO: 125) and has an IgE signal sequence (SEQ ID NO: 176) at the N-terminus of human IL-15.

[0339] HER1t was constructed by connecting domain III of human HER1 (SEQ ID NO: 98) to amino acids 1-21 of domain 4 of human HER1 (SEQ ID NO: 100), with an Igκ signal sequence (SEQ ID NO: 169 or SEQ ID NO: 170) at the N-terminus of domain III. The resulting sequence was connected to the human CD28 transmembrane domain (SEQ ID NO: 101) via a Gly-Ser-rich linker peptide (SEQ ID NO: 102).

[0340] To explore the effect of gene / element order on expression and function, three tricistronic polynucleotide expression cassettes (Cassettes 1-3) were generated. The 5' to 3' order of elements in each expression cassette is as follows: Cassette 1: CD19CAR-F2A-mbIL15-T2A-HER1t, Cassette 2: mbIL15-T2A-HER1t-F2A-CD19CAR, and Cassette 3: HER1t-T2A-mbIL15-F2A-CD19CAR. The polynucleotide sequence of each expression cassette is shown in Table 10.

[0341] The corresponding theoretical polypeptide translation products of each expression cassette, without considering N-terminal signal sequence cleavage or ribosomal skipping at each F2A and T2A site, are shown in Table 11.

[0342] Six recombinant nucleic acid Sleeping Beauty transposon plasmids incorporating the aforementioned expression cassettes were generated. In each plasmid, one of cassettes 1-3 and a suitable transcriptional regulatory element was flanked by a pair of inverted terminal repeats (ITRs) recognized by the Sleeping Beauty transposase SB11. For each expression cassette, two pairs of ITR pair α and ITR pair β were evaluated. The six resulting transposon plasmids are summarized in Table 14.

[0343] [Table 14]

[0344] For control purposes, two additional transposon plasmids were prepared: plasmid DP1 encoding CD19CAR, and plasmid DP2, which contains an expression cassette encoding mbIL15-T2A-HER1t from the N-terminus to the Cmatt tan. Plasmids DP1 and DP2, when combined in a 1:1 ratio, are referred to herein as "dTp control."

[0345] 6.2 Example 2: Generation and evaluation of T cells co-expressing CD19CAR, mbIL15, and HER1t This example describes the generation and evaluation of T cells co-expressing CD19CAR, mbIL15, and HER1t from the plasmids described in Example 1.

[0346] 6.2.1 Materials and Methods 6.2.1.1 Cell lines K562-derived activated proliferating cells (AaPCs), designated as clone 9, expressing CD64, CD86, CD137L, and truncated CD19 (e.g., as described in Denman et al., PLoS One. 2012;7(1):e30264, the contents of which are incorporated herein by reference in their entirety), were used ex vivo for expansion of genetically modified T cells. The target cell line for the cytotoxicity assay was CD19 + (NALM-6, Daudi, CD19-EL4, and CD19-negative (parental EL4) tumor cell lines were obtained from the American Type Culture Collection (Manassas, VA) (or as described, for example, in Singh et al., PLoS One. 2013;8(5):e64138, the contents of which are incorporated herein by reference in their entirety). Cells were routinely cultured in R10 (RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS, Hyclone / GE Healthcare, Logan, UT) and 1% Glutamax-100 (ThermoFisher Scientific, Waltham, MA)). Cells were cultured in standard conditions at 37° C. and 5% CO2. Cells were tested and found to be negative for mycoplasma. Cell line identity was confirmed by short tandem repeat DNA fingerprinting.

[0347] 6.2.1.2 Normal donor human T cells Peripheral blood or leukapheresis transfusion products were obtained from normal donors (Key Biologics, Memphis, TN). A T cell enriched starting product was used. The apheresis product was diluted using CliniMACS® PBS / EDTA buffer containing 0.5% (v / v) HSA and a platelet depletion step was performed by centrifugation at 400×g for 10 min at room temperature (RT) followed by resuspension in the same buffer. According to the manufacturer's protocol, CD4 and CD8 specific CliniMACS (cryopreserved) microbeads (CD4 GMP MicroBeads #170-076-702, CD8 GMP MicroBeads #170-076-703, Miltenyi) were incubated with the cells for 30 min at room temperature under mixed conditions to enrich the starting product for T cells, which were then subjected to paramagnetic selection in CliniMACS Plus. Live / dead cells were enumerated with a Cellometer instrument (Nexcelom Bioscience, Lawrence, MA). Isolated T cells were cryopreserved in CryoStor CS10 and stored in the vapor phase of a liquid nitrogen tank.

[0348] 6.2.1.3 Generation of RPM CD19CAR-mbIL15-HER1t T cells using the SB line To generate the CAR-T cells described in this example, a Nucleofector (cryopreservation) 2b device (Lonza, Basel, Switzerland) was used to transfer dTp control or plasmids A-F into the T cell enriched starting product as described in Example 1. Plasmid TA encoding the SB11 transposase was co-transfected in each case of transposon transfection to allow stable genetic integration of the transposon. A schematic of the gene transfer process for both double transposition (using dTp control) and single transposition (using plasmids A-F) is shown in Figure 2.

[0349] The day before electroporation, cryopreserved CD3 enriched cells were thawed in R10, washed and resuspended in R10 and placed in a 37° C. / 5% CO2 incubator overnight. Electroporation details for each test article are as follows:

[0350] Mock CD3 cells (no DNA, also referred to herein as "negative control"): Resting cells were harvested, spun down, and resuspended in device-specific Nucleofector buffer (Human T Cell Nucleofector Kit, Lonza) without any DNA plasmids.

[0351] dTp control RPM CD19CAR-mbIL15-HER1t T cells: Resting cells were harvested, spun down and resuspended in Nucleofector buffer containing transposon DNA (dTp control) and transposase DNA (plasmid TA, encoding SB11 transposase) at a final transposon:transposase ratio of 3:1.

[0352] sTp RPM CD19CAR-mbIL15-HER1t T cells: Resting cells were harvested, spun down, and resuspended in Nucleofector buffer containing transposon DNA (one of plasmids A-F) and transposase DNA (plasmid TA) at a final transposon:transposase ratio of 3:1.

[0353] Immediately after electrotransfer, the contents from each cuvette were resuspended and transferred to R10 medium containing DNase for 1-2 hours of incubation in a 37°C / 5% CO2 incubator. A full medium change was then performed with R10 medium and the cells were placed in a 37°C / 5% CO2 incubator overnight. Within 24 hours (and at least 16 hours) after electrotransfer (day 1), cells were harvested from the culture and sampled by flow cytometry to determine cell surface expression of CD19CAR, mbIL15, and HER1t.

[0354] On day 1, transfected T cells were stimulated with γ-irradiated (100 Gy) K562-AaPC clone 9 at a 1:1 T cell / AaPC ratio. Additional γ-irradiated AaPC ​​clone 9 was added at the same ratio every 7-10 days. Soluble recombinant human IL-21 (catalog no. 34-8219-85, eBioscience, San Diego, CA) was added at a concentration of 30 ng / mL starting the day after electroporation and replenished three times a week for a 7-10 day stimulation cycle marked by the addition of AaPC ​​(each such stimulation cycle referred to as "Stim"). T cells were enumerated at the end of each Stim and viable cells were counted based on AOPI exclusion using a Cellometer automated cell counter. Expression of T cell markers, CD19CAR, mbIL15, and HER1t, was assessed using flow cytometry every 7-10 days. Expansion of unwanted NK cells in the cultures was addressed by depletion (positive selection using CD56 microbeads, Miltenyi) according to the manufacturer's instructions. At the end of Stim 1, 2, 3, and 4, total cells, CD3 + , CD19CAR + , and HER1t + T cell expansion was determined.

[0355] 6.2.1.4 Flow cytometry Max 1×10 6Cells were stained with human-specific fluorochrome-conjugated antibodies. Staining of cell surface markers on samples and corresponding controls was performed first by performing an Fc receptor blocking step and incubating with 50% mouse serum (Jackson ImmunoResearch, PA) in FACS buffer (PBS, 2% FBS, 0.1% sodium azide) for 10 minutes at 4°C to reduce background staining. Immunostaining was performed by adding 100 μL of antibody master mix of the antibody combinations listed in Table 15 diluted in Brilliant Stain Buffer (BD Biosciences). Briefly, CD19CAR expression was detected using an Alexa Fluor (cryopreserved) (AF)488-conjugated anti-idiotypic antibody (clone number 136.20.1) specific for the anti-CD19 portion of CD19CAR (e.g., as described in Jena et al., PLoS. 2013;8(3):e57838, the contents of which are incorporated herein by reference in their entirety). CD19CAR anti-idiotype antibody was conjugated to AF-488 fluorophore by Invitrogen / Thermo Fisher Scientific (Waltham, MA). HER1t molecules were detected using fluorescently conjugated cetuximab antibody. Fluorescently conjugated cetuximab reagent was commercially purchased Erbitux conjugated to AF-647 by Invitrogen / Thermo Fisher Scientific. Fluorescently conjugated antibodies used included CD3 (clone SK7), IL-15 (34559), CD45 (clone HI30), and CD19-CAR idiotype (clone 136.20.1) (Table 15).

[0356] [Table 15]

[0357] Master mixes containing the antibody combinations in Table 15 were added sequentially (CD19CAR, mbIL15, followed by the remaining antibody cocktail) and incubated for 30 minutes at 4°C. Cells were washed with FACS buffer and then incubated with fixable viability stain-620 viability dye (1:1000 in PBS, BD Biosciences) for 10 minutes at 4°C, followed by washing with FACS buffer. Data were acquired using an LSR Fortessa (BD Biosciences) with FACSDiva software (v.8.0.1, BD Biosciences) and analyzed with FlowJo software (version 10.4.2, TreeStar, Ashland, OR). Transgene expression was measured for gated cell events, singlets, viable events, and CD3, unless otherwise stated. + The cells were evaluated.

[0358] 6.2.1.5 Western blot analysis Ex vivo expanded CD19CAR modified T cells were centrifuged and the pellet was lysed in RIPA buffer containing protease inhibitors (Complete Mini, Roche). Lysates were incubated at 4 °C for 20 min and the supernatants were stored at -20 °C. A bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, 23227) was performed to determine the total protein concentration of the lysates. Western blots were performed on a Wes 2010 Western blot platform (ProteinSimple, Wes 2010) according to the manufacturer's instructions. For each sample, 0.1–0.2 μg / mL of protein lysate was mixed with 5× fluorescence master mix (ProteinSimple, DM-002), heat denatured, chilled on ice and loaded onto the cartridge (ProteinSimple, SM-W004). For detection of CD19CAR protein, mouse anti-human CD247 (BD Biosciences, 551033) primary antibody and HRP goat anti-mouse (ProteinSimple, DM-002) secondary antibody were used. Jurkat cells expressing CD19 CAR were used as a positive control. For detection of mbIL15 chimeric protein, primary antibody goat anti-human IL-15 (R&D, AF315) and secondary antibody HRP anti-goat (ProteinSimple, 043-552-2) were used. Recombinant human IL-15 protein (R&D, 247-ILB) was loaded as a positive control. For detection of HER1t chimeric protein, primary antibody mouse anti-human EGFR (Sigma, AMAB90819-100μL) and secondary antibody HRP anti-mouse (ProteinSimple, DM-002) were used. Human EGFR protein (Biosystems Acro, EGR-H5252-100 μg) was used as a positive control.

[0359] 6.2.1.6 Chromium release assay The antigen-specific cytotoxicity of ex vivo expanded CD19-specific T cells generated using dTp control, plasmid A, and plasmid D was investigated by radiolabeling ( 51 Cr) Determined by lysis of target cells. CD19 + (NALM-6, Daudi, CD19-EL4) and CD19-negative (EL4) tumor cell lines were used as targets. T cells and radiolabeled target cells were co-incubated in triplicate and lysis was determined by measuring radioactivity in the supernatant at the end of a 4-h incubation. Chromium release was detected using a TopCount NXT (Perkin Elmer) and specific lysis was calculated as follows:

[0360]

number

[0361] Media and Triton-X 100-treated target cells served as background and maximum lysis controls, respectively. Mean ± SD of dTp control (N=6), plasmid A (N=4), and plasmid D (N=1) lysis at each E:T ratio was calculated.

[0362] 6.2.1.7 Antibody-dependent cytotoxicity (ADCC) ADCC of CD19-specific T cells expressing mbIL15-HER1t was determined by a modified 4-h chromium release assay, whereby T cells (with specific antibody treatment) served as target cells and ex vivo activated, expanded NK cells expressing Fc receptors were used as effector cells. A range of five different effector-to-target (E:T) ratios (40:1, 20:1, 10:1, 5:1, and 2.5:1) were tested to determine the effect of CD19-specific T cells expressing mbIL15-HER1t on ADCC. 51Measurement of the amount of target lysis was established by detection of Cr release. Ex vivo expanded (Stim4)CD19CAR-mbIL15-HER1t T cells were incubated with the HER1t specific antibody cetuximab (Imclone LLC, NDC 66733-948-23) or the non-specific (irrelevant) antibody rituximab (Biogen Inc. and Genentech USA Inc., NDC 50242-051-21) at 20 μg / mL for 20-30 min at room temperature and these T cells were used as targets. NALM-6 and K562 cell lines were used as negative and positive controls (without antibody treatment), respectively, to evaluate the cytolytic activity of NK cells. Target cells treated with medium only or Triton X-100 (Sigma) were used as controls for spontaneous and maximum lysis, respectively. Percentage (%) 51 Cr dissolution was calculated as follows:

[0363]

number

[0364] Percent lysis data were normalized to the maximum cell lysis observed by NK cells. Means ± SD were calculated for dTp control (N=6), Plasmid A (N=4), and Plasmid D (N=1).

[0365] 6.2.1.8 Quantitative Droplet Digital PCR (ddPCR) to Determine Transgene Copy Number The ddPCR method was used to determine the presence and quantification of CD19CAR, mbIL15, and HER1t average transgene integration events per cell in genetically modified T cells. Ex vivo expanded (Stim4)CD19CAR-mbIL15-HER1t T cells transfected with double transposon control or test plasmids (DtP control or plasmids A-F, respectively), mock transfected CD3 (negative control without DNA), CD19CAR + Jurkat cells (positive control for CD19CAR), mbIL15 +Jurkat cells (positive control for mbIL15), and CD19CAR + HER1t +Genomic DNA (gDNA) from T cells (positive control for HER1t) was isolated using a commercially available kit (Qiagen). Primer / probe sequences were designed to be specific for the CD19CAR, mbIL15, and HER1t transgenes. Target primers / probes were synthesized by Bio-Rad system (Bio-Rad) using FAM-labeled probes. All samples were duplexed with the specific human endogenous reference gene EIF2C1 using a HEX-labeled probe (Bio-Rad). PCR droplets were generated with DG8 cartridges (Bio-Rad) using a QX-100 droplet generator according to the manufacturer's protocol, and 20 μL of each PCR mixture was dispensed into droplets of approximately 20,000 nanoliter size. PCR droplets were transferred to a 96-well PCR plate and sealed with foil. PCR was performed using a Bio-Rad C1000 thermal cycler [95°C (10 min); 40 cycles of 94°C (30 s), 58°C (30 s), and 98°C (10 min); 12°C (indefinite)]. DNA copy number was assessed using a QX-100 Digital Droplet PCR System (Bio-Rad). All samples were run in triplicate. After the reaction in the thermocycler was completed, the PCR plate was transferred to a QX200™ Droplet Digital™ PCR System reader to acquire data. Data was analyzed using QuantaSoft™ software (version 1.7.4, Bio-Rad). To determine the transgene copy number, the ratio of the target (CD19CAR, mbIL15, and HER1t) to the reference gene (EIF2C1) was multiplied by 2, since each cell contains two copies of the reference EIF2C1 gene. Copy number variation (CNV) settings were utilized in the software program, with reference genes set to 2 copies / cell (see, e.g., Belgrader et al., Clinical Chemistry, 2013;59(6):991-994, and Hindson et al., Anal Chem. 2011;83:8604-8610, the contents of each of which are incorporated herein by reference in their entireties).In the QuantaSoft™ software, copy number is automatically determined by calculating the ratio of the target molecule concentration to the reference molecule concentration multiplied by the copy number of the reference species in the genome.

[0366] 6.2.1.9 Statistical analysis Statistical tests are described with each statistic reported. Post-hoc analyses were performed to compare differences between treatment groups and are reported with each statistical result. Errors are reported as standard deviations (SD). Statistical analyses were performed using GraphPad Prism (version 8) software. P<0.05 was considered statistically significant.

[0367] 6.2.2 Genetic Modification, Expression Characterization, and Expansion of CAR-T Cells Co-Expressing CD19CAR, mbIL15, and HER1t Donor T cell enriched starting products were transfected with either no transposon plasmid (negative control), dTp control, or plasmids A-F. RPM CD19CAR-mbIL15HER1t T cells were generated from three donors by electroporation using the SB system, and the resulting transgenic subpopulations present in the RPM T cell product (CD19CAR + -mbIL15-HER1t + , CD19CAR + mbIL15-HER1t 陰性 , CD19CAR 陰性 -mbIL15-HER1t + , CD19CAR 陰性 -mbIL15-HER1t 陰性 ) was performed one day after transfection (Table 16).

[0368] [Table 16]

[0369] On day 1, each of the RPM CAR-T cell groups had comparable mean viability (62%-64%) (Table 16 and Figure 3A) and a mean CD3 +The frequencies of CD19CAR expression in sTp variants were shown in Table 16 and Figure 3B. With regard to the evaluation of individual transgene expression, Plasmid A (31% ± 13%), Plasmid B (28% ± 15%), and Plasmid D (28% ± 17%) yielded the highest CD19CAR expression among sTp variants, with approximately 1.5-fold greater expression than the dTp control (20% ± 15%), corresponding to the CD19CAR transgene at position 1 (mostly N-terminus) or 3 (mostly C-terminus) (Table 16 and Figure 3C). Plasmid B (24% ± 9%) and Plasmid E (20% ± 11%) showed the highest expression of mbIL15, followed by Plasmid A (13% ± 5%) and Plasmid D (16% ± 12%), which were higher than the 9% ± 8% expression observed by the dTp control modified T cells, corresponding to the mbIL15 transgene at position 1, followed by intermediate expression when it was at position 2 (intermediate position) (Table 16 and Figure 3D). Plasmid A, Plasmid D, and Plasmid F had the highest HER1t expression (30% ± 11%, 29% ± 15%, and 34% ± 5%, respectively), which was approximately two-fold greater expression than the dTp control (13% ± 13%) and corresponded to HER1t at positions 1 or 3 (Table 16 and Figure 3E).

[0370] Cells from donor A were expanded ex vivo by four rounds of recurrent stimulation on K562-AaPC clone 9. Transgene co-expression was assessed via two-parameter flow plots at day 1 and at the end of Stim1, 2, 3, and 4, as shown in Figures 4A-4F, 5A-5F, 6A-6F, 7A-7F, 8A-8F, 9A-9F, and 10A-10F. On day 1, dTp control modified T cells were transduced with CD19CAR + HER1t + (5%) / CD19CAR + mbIL15 + (3%) small population of T cells (Figures 4D and 4E, respectively), as well as CD19CAR + HER1t 陰性We observed that an approximately two-fold larger population of T cells (9%) displayed the standard heterologous transgene expression pattern (Figure 4D). Low levels of co-expression were observed for HER1t and mbIL15 (2%) (Figure 4F). Plasmid A modified T cells were significantly more potent than CD19CAR and HER1t (17%) and HER1t (8%). + mbIL15 + T cells (Figures 5D and 5F, respectively) and 12% HER1t + mbIL15 陰性 ( Fig. 5F ), and 8% CD19CAR + mbIL15 + Plasmid B-modified T cells showed poor HER1t expression (21% CD19CAR + HER1t 陰性 and 5% CD19CAR + HER1t + ) (Figures 6D and 11C), whereas mbIL15 (16% of CD19CAR + mbIL15 + ) expression (Figures 6E and 11B). Plasmid C modified T cells showed co-expression of CD19CAR and HER1t (13%) (Figure 7D), but lower HER1t expression compared to Plasmid A. + mbIL15 + Plasmid D-modified T cells showed 27% co-expression of CD19CAR (Figure 7F). + HER1t + Subset and 8% CD19CAR + HER1t 陰性 MbIL15 also showed good expression, with 18% of CD19CAR + mbIL15 + Co-expression of HER1t and mbIL15, as well as HER1t expression greater than mbIL15 (13% of HER1t + mbIL15 陰性 and 16% HER1t + mbIL15 + ) there was some heterogeneity between the plasmid B modified T cells and the plasmid E modified T cells (Figure 8F). Similar to the plasmid B modified T cells, the plasmid E modified T cells showed poor HER1t expression (20% CD19CAR +HER1t 陰性 and 5% CD19CAR + HER1t + ) (Figures 9D and 11C), whereas mbIL15 (16% of CD19CAR + mbIL15 + ) (Figures 9E and 11B). Similar to plasmid C-modified T cells, plasmid F-modified T cells showed improved expression of CD19CAR and HER1t (25%) (Figure 10D), as well as 13% HER1t + mbIL15 + Overall, the transgene expression pattern on day 1 in RPM T cells showed the most favorable CD19CAR / HER1t co-expression and total mbIL15 expression in Plasmid A and Plasmid D, followed by Plasmid F.

[0371] Stim4 ex vivo expanded T cells obtained CAR expression over 90% in all treatments. The highest mbIL15 expression was observed in plasmid A and plasmid D modified cells (66% and 72%, respectively) compared to 63% in dTp control modified T cells (Figure 4C) (Figures 5C and 8C, and Figure 11B, respectively). Furthermore, the highest total HER1t expression was observed only in plasmid A and plasmid D modified cells (95%, respectively) (Figures 5B, 8B, and 11C), which exceeded the dTp control (78%) (Figures 4B and 11C) and was crucially superior to the other sTp variants, which all showed expression below 44% (Figures 6B, 7B, 9B, 10B, and 11C).

[0372] Notably, ex vivo expanded CD19CAR in Stim4 was expressed in plasmid A modified cells (94%) and plasmid D modified cells (94%). + HER1t + Not only was co-expression the highest, but furthermore, expression levels of CD19CAR and HER1t were also highly correlated, with a uniform CAR+HER1t + This resulted in a pattern of diffuse CAR expression exhibited by dTp control modified cells (Figures 5D and 8D). + HER1t+ Similarly, the CAR expression levels in plasmid A- and D-modified cells were significantly higher than in the control population (Figure 4D). + mbIL15 + The expression pattern was highly correlated and uniform, in contrast to the diffuse pattern observed in dTp control modified cells (Figures 5E, 8E, and 4E, respectively).

[0373] Protein expression confirmation was performed on cell lysates from Stim4 ex vivo expanded CD19CAR-mbIL15-HER1t T cells by Western blot. Cells were prepared, protein transferred and probed with anti-human CD247 for detection of CD19CAR protein on modified cells (Figure 12A), anti-human IL-15 for detection of mbIL15 (Figure 12B), and anti-human EGFR for detection of HER1t (Figure 12C). Secondary HRP antibodies with appropriate specificity were used for detection. Jurkat cells expressing CD19CAR were used as a positive control for CD19CAR detection. Recombinant human IL-15 (rhIL-15) and no DNA (negative control) T cells served as positive and negative controls, respectively, for detection of chimeric IL-15. Recombinant human EGFR was used as a positive control for detection of truncated EGFR (tEGFR).

[0374] CD19CAR expression was confirmed by Western blot analysis of CD3ζ using anti-CD3ζ antibody. As shown in Figure 12A, detection of endogenous CD3ζ band (approximately 16 kDa) was observed in all T cell samples. The band / band at approximately 60 kDa represents the chimeric CD3ζ protein of CD19-specific CAR. Detection of control rhIL-15 occurred at the expected approximately 15 kDa, and the chimeric mbIL15 band was observed at approximately 140 kDa (Figure 12B). HER1t (truncated EGFR, tEGFR) expression was observed in modified T cells at approximately 50 kDa, and full-length EGFR was detected in rhEGFR at approximately 190 kDa (Figure 12C).

[0375] Numerical expansion was assessed in Donor A for all transposon variants. T cell enriched starting products were thawed and allowed to rest overnight. Cells were electroporated using Amaxa Nucleofector solution along with dTp controls and compared to Plasmids A-F for Donor A. The next day cells were stimulated with γ-irradiated (100 Gy) K562-AaPC clone 9. Additional recurrent stimulations (Stim) were performed every 7-10 days. T cells were enumerated on day 1 and at the end of each Stim, and viable cells were counted based on AOPI exclusion using a Cellometer automated cell counter. Overall, all cultures achieved numerical expansion. CD19CAR-specific expansion was approximately 0.5-1 log greater than the dTp controls for all sTp variants (Figure 13A). mbIL15-specific expansion was approximately 0.5-1 log greater than the dTp control for all sTp variants except for Plasmid E, which had expansion comparable to the dTp control (Figure 13B). HER1t-specific expansion varied. Plasmid B and Plasmid E showed the lowest expansion of HER1t+ T cells, Plasmid C and Plasmid F showed expansion comparable to the dTp control, and Plasmid A and Plasmid D showed the greatest numerical expansion (Figure 13C).

[0376] This example shows that Plasmid A and Plasmid D are capable of achieving the primary objectives of genetic modification of T cells with the CD19CAR-mbIL15-HER1t tricistronic transposon plasmid, i.e., redirecting antigen specificity to CD19CAR, mbIL15, and HER1t. + These results show that the plasmid A and plasmid D single transposon constructs with the element order CD19CAR-F2A-mbIL15-T2A-HER1t best meet the desired criteria, while the ....

[0377] 6.2.3 Functional characterization of CAR-T cells co-expressing CD19CAR, mbIL15, and HER1t Assays were performed to evaluate the functional characteristics of CAR-T cells co-expressing CD19CAR, mbIL15, and HER1t.

[0378] 6.2.3.1 Specificity of CD19-directed cytotoxicity and cytokine expression using CAR-T cells expressing CD19CAR, mbIL15, and HER1t Cytotoxicity assays were performed to detect CD19 + Showed specificity in targeting tumor cells. CD19 + Specificity for tumor targets was determined using CD19-expressing tumor cell lines (NALM-6, Daudi β2M, and engineered CD19EL-4) and CD19 陰性 This was demonstrated by comparing activity with that of the parental EL-4 cell line. Cytotoxicity assays tested E:T ratios ranging from 20:1 to 1.25:1 in a standard 4-hour chromium release assay. CD19CAR-mbIL15-HER1t T cells transfected with plasmids A-F showed specific lysis of approximately 50% of all CD19+ targets at the lowest E:T, comparable to dTp control cells (Figures 14A-14H). 陰性 Target lysis was minimal at low E:T. In summary, modification of T cells with plasmids A–F, which result in coexpression of single transposon-derived transgenes, did not alter the cytotoxic function of CD19CAR-mbIL15-HER1t T cells compared to cells modified with the dTp control.

[0379] 6.2.3.2 HER1t-Mediated Depletion of CD19CAR-mbIL15-HER1t T Cells via ADCC Co-expression of mbIL15 and CD19CAR with HER1t on the cell surface and infusion of mbIL15 +HER1t was included in the tricistronic design to provide a mechanism for selectively depleting T cells. HER1t-expressing cells can be eliminated by administration of cetuximab, a clinically available monoclonal antibody that binds to HER1t and mediates antibody-dependent cellular cytotoxicity (ADCC). In vitro evaluation was performed to confirm the ability of cetuximab to induce ADCC against ex vivo expanded CD19CAR-mbIL15-HER1t T cells. Genetically modified T cells served as targets in this assay, which was a standard 4-hour chromium release assay in the presence of cetuximab (anti-HER1t antibody) or rituximab (anti-CD20 antibody, negative control), using Fc receptor-expressing NK cells as effectors. As shown in Figure 15, the addition of cetuximab caused depletion of target HER1t-modified T cells generated with dTp control, Plasmid A, Plasmid C, Plasmid D, and Plasmid F. CD19CAR-mbIL15-HER1t T cells generated with plasmid A and plasmid D showed the highest levels of selective depletion (approximately 60% and 50%, respectively). Cetuximab was significantly more potent than the negative control (HER1t 陰性 ) failed to demonstrate cell lysis, confirming a HER1t-specific mechanism of action.

[0380] These data support the use of cetuximab to deplete CD19CAR-mbIL15-HER1t T cells generated using Plasmid A and Plasmid D in the event of adverse clinical effects necessitating a depletion strategy.

[0381] 6.2.3.3 Stable integration of CD19CAR, mbIL15, and HER1t transgenes following ex vivo expansion of SB-modified CD19CAR-mbIL15-HER1t T cells The copy numbers of CD19CAR, mbIL15, and HER1t transgenes in ex vivo expanded CD19CAR-mbIL15-HER1t T cells were examined using ddPCR and primer / probe sets specific for CD19CAR, mbIL15, and HER1t. The results are shown in FIG. 16. Copy numbers were normalized to the human reference gene EIF2C1, known to be present at 2 copies / cell. It was observed that the dTp controls had different integration levels between CD19CAR and each of mbIL15 and HER1t (about 2.5 copies per cell for CD19CAR and about 8 copies per cell for mbIL15 and HER1t). T cells generated with plasmid C and plasmid F showed more than 10 transgene copies per cell. Cells generated with plasmid A, plasmid D, and plasmid E showed an average copy number of about 5 per cell, while cells generated with plasmid B showed an average copy number of about 7 per cell. Positive control T cells (grown on AaPC) showed transgene insertion at an average of approximately 1 copy per cell.

[0382] In summary, based on an analysis of the number of transposon insertions into the primary human T cell genome of cells produced under RPM, such cells underwent stable integration of the transgene, with CD19-mbIL15-HER1t T cells generated by Plasmid A, Plasmid D, and Plasmid E exhibiting the most favorable (low) integration values ​​compared to other sTp variants and the dTp control. In addition, T cells generated by all sTp variants exhibited much more consistent integration values ​​across all three transgenes than T cells generated by the dTp control.

[0383] 6.3 Example 3: Multi-donor evaluation of candidate tricistronic sTp SB DNA plasmids Evaluation of the sTp plasmids in Example 2 identified Plasmid A and Plasmid D as candidates for further testing based on (i) favorable co-expression of the transgenes at day 1 and Stim4 as detected by flow cytometry, (ii) overall transgene expression Stim4 as detected by Western blot, (iii) acceptable transgene-specific numerical expansion, (iv) unaffected cytotoxicity, and (v) favorable selective elimination. This example describes the continued evaluation of candidate Plasmid A in additional donors. Plasmid D data from a single donor is included for reference and is comparable to Plasmid A since the transgene order is the same.

[0384] 6.3.1 Materials and Methods Materials and methods were as described in section 6.2.1 except where indicated.

[0385] 6.3.2 Genetic Modification, Expression Characterization, and Expansion of CAR-T Cells Co-Expressing CD19CAR, mbIL15, and HER1t Similar to Example 2, T cell enrichment products were electroporated with dTp control, Plasmid A, and Plasmid D and expanded ex vivo by co-culture on irradiated clone 9 AaPCs, and RPM T cells (day 1) and Stim4 expanded cells were evaluated. Growth kinetics and transgene-specific expansion of T cells generated using dTp control (n=10, day 1; n=5, Stim1; n=7, Stim2; n=6, Stim3; n=5, Stim4, FIG. 17A), Plasmid A (n=8, day 1; n=3, Stim1; n=4, Stim2; n=4, Stim3; n=4, Stim4, FIG. 17B), and Plasmid D (n=7, day 1; n=2, Stim1; n=2, Stim2; n=1, Stim3; n=1, Stim4, FIG. 17C) were approximately 10 11 The results were comparable across cell treatments.

[0386] Similarly, T cell enriched products were electroporated with dTp control (n=6, day 1; n=3, Stim4) (FIG. 18A), plasmid A (n=3, day 1; n=3, Stim4) (FIG. 18B), and plasmid D (n=1, day 1; n=1, Stim4) (FIG. 18C) and expanded ex vivo by co-culture on irradiated clone 9 AaPCs. Assessment of CD19CAR / HER1t co-expression in RPM T cells on day 1 demonstrated that cells modified with plasmid A or plasmid D significantly up-regulated the target CD19CAR compared to dTp control modified cells. + HER1t + At the end of Stim4, both plasmid A and plasmid D modified cells were shown to have higher frequencies of the target CD19CAR T cell population compared to dTp control modified cells (7% ± 9%, 27% ± 0%, and 2% ± 2%, respectively). + HER1t + The donors had higher frequencies of T cell populations (67%±27%, 94%±0%, 50%±34%, respectively). Additional donor evaluation of the dTp control and Plasmid A supports the observation in Example 2 that transgene co-expression is improved by the use of Plasmid A and Plasmid D (which share the same transgene order) compared to the dTp control.

[0387] 6.3.3 Functional characterization of CAR-T cells co-expressing CD19CAR, mbIL15, and HER1t Assays were performed to evaluate the functional characteristics of CAR-T cells co-expressing CD19CAR, mbIL15, and HER1t.

[0388] 6.3.3.1 Specificity of CD19-directed cytotoxicity and cytokine expression using CAR-T cells expressing CD19CAR, mbIL15, and HER1t Cytotoxicity was assessed in a standard 4-hour chromium release assay in additional donors for ex vivo expanded CD19CAR-mbIL15-HER1t T cells generated with dTp control (n=6), plasmid A (n=4), and plasmid D (n=1), as in section 6.2.3.1. + Cytotoxicity of the target cell lines was comparable across the three conditions, with approximately 40% specific lysis observed for the dTp control (Figure 19A) and Plasmid A (Figure 19B) and approximately 50% specific lysis observed for Plasmid D (Figure 19C) at an E:T ratio of 1.25:1. CD19 陰性 Cell lysis was negligible. In summary, these data indicate that Plasmid A and Plasmid D do not alter cytotoxic potential, further supporting the observations in Section 6.2.3.1.

[0389] 6.3.3.2 HER1t-Mediated Depletion of CD19CAR-mbIL15-HER1t T Cells via ADCC Similar to section 6.2.3.2, selective elimination of CD19CAR-mbIL15-HER1t T cells via ADCC was evaluated in additional donors for ex vivo expanded CD19CAR-mbIL15-HER1t T cells generated with dTp control (n=6), Plasmid A (n=4), and Plasmid D (n=1). For all three conditions, cetuximab treatment resulted in approximately 50% lysis of target CD19CAR-mbIL15-HER1t T cells by effector NK cells (Figure 20). These data provide further support to the data in section 6.2.3.2 showing that Plasmid A and Plasmid D generate CD19CAR-mbIL15-HER1t T cells that can be selectively depleted via ADCC using cetuximab.

[0390] 6.3.3.3 Stable integration of CD19CAR, mbIL15, and HER1t transgenes following ex vivo expansion of SB-modified CD19CAR-mbIL15-HER1t T cells Similar to section 6.2.3.3, ex vivo expanded Stim4 CD19CAR-mbIL15-HER1t T cells generated using dTp control (n=7), Plasmid A (n=5), or Plasmid D (n=1) were assessed for transgene copy number using ddPCR and primer / probe sets specific for CD19CAR, mbIL15, and HER1t, as shown in Figure 21. Cells generated with the dTp control showed an average of about 3 copies / cell for CD19CAR, about 11 copies / cell for mbIL15, and about 11 copies / cell for HER1t. Cells generated with Plasmid A had an average of about 6 copies / cell for the three transgenes, and cells generated with Plasmid D had an average of about 5 copies / cell for the three transgenes.

[0391] In summary, these data confirm the observations from section 6.2.3.3, where Plasmid A and Plasmid D each generated CD19CAR-mbIL15-HER1t T cells with nearly identical integration numbers for all three transgenes, whereas the dTp control generated cells with substantially different integration numbers between CD19CAR on the one hand and mbIL15 and HER1t on the other, indicating that mbIL15 and HER1t are integrated at substantially higher levels.

[0392] 6.4 Example 4: In vivo generation and evaluation of RPM T cells co-expressing CD19CAR, mbIL15, and HER1t This example describes the in vivo generation and evaluation of RPM T cells co-expressing CD19CAR, mbIL15, and HER1t from dTp control or Plasmid A.

[0393] 6.4.1 Materials and Methods 6.4.1.1 Cell lines The human tumor cell line NALM-6 / fLUC was cultured at MD Anderson Cancer Center (MDACC, Houston, TX) using parental pre-B cell CD19 +The NALM-6 cell line (American Type Culture Collection (ATCC, Manassas, VA)) was generated (or as described, for example, in Singh et al., Cancer Res. 2011;71(10):3516-3527, the contents of which are incorporated herein by reference in their entirety). These tumor cells co-express firefly luciferase (fLUC) for non-invasive bioluminescence imaging (BLI) and enhanced green fluorescent protein (EGFP) for fluorescent imaging. Cells were routinely cultured in RPMI 1640 or Hyclone:R10 medium containing 10% FBS (Hyclone / GE Healthcare, Logan, UT) and 1% Glutamax-100 (ThermoFisher Scientific, Waltham, MA). Cells were cultured in routine conditions at 37° C. and 5% CO2. Cells were tested and found to be negative for mycoplasma. The identity of the cell lines was confirmed by short tandem repeat DNA fingerprinting.

[0394] 6.4.1.2 Normal donor human T cells Peripheral blood or leukapheresis products were obtained from normal donors (Key Biologics, Memphis, Tenn.). Multiple collections were obtained from the same donor. Apheresis products were split to allow for testing of two starting cell products for the manufacture of RPM T cells.

[0395] A portion of the apheresis was processed for isolation of PBMCs using a Sepax S-100 Cell Separation System (BioSafe, Newark, DE). Live / dead cells were enumerated with a Cellometer instrument (Nexcelom Bioscience, Lawrence, MA). Isolated PBMCs were cryopreserved in a CryoStor CS10 (Biolife Solutions, Bothell, WA, or equivalent) and stored in the vapor phase of a liquid nitrogen tank.

[0396] The other part of the apheresis product, preparation of the T cell enriched starting product (for the CD3 treatment group), was diluted using CliniMACS® PBS / EDTA buffer containing 0.5% (v / v) HSA, and a platelet depletion step was performed by centrifugation at 400×g for 10 min at room temperature (RT), followed by resuspension in the same buffer. Both CD4 and CD8 specific CliniMACS® microbeads were incubated with the cells under mixed conditions for 30 min at room temperature, and paramagnetic selection was performed with CliniMACS Plus to enrich the T cell starting product. Live / dead cells were enumerated with a Cellometer instrument (Nexcelom Bioscience, Lawrence, MA). Isolated T cells were cryopreserved in CryoStor CS10 and stored in the vapor phase of a liquid nitrogen tank.

[0397] 6.4.1.3 Generation of RPM CD19CAR-mbIL15-HER1t T cells using the SB line To generate the test article population of RPM CD19CAR-mbIL15-HER1t T cells evaluated in this study, either PBMC or T cell enriched starting product was used and gene transfer was performed using either dTp control or Plasmid A using a Nucleofector™ 2b device (Lonza, Basel, Switzerland), respectively, as described in Example 1. Details of the generation of each test article are as follows:

[0398] Mock PBMCs: The day before electroporation, cryopreserved PBMCs were thawed in RPMI 1640 medium (phenol red-free medium (Hyclone), 10% FBS, and 1% Glutamax-100 (R10)), washed with R10, resuspended, and placed in a 37°C / 5% CO2 incubator overnight. Resting cells were harvested, spun down, and resuspended in Nucleofector buffer (Human T Cell Nucleofector Kit, Lonza) without any transposon or transposase DNA plasmids.

[0399] Mock CD3: Cryopreserved CD3 enriched cells were thawed and processed as described above for mock PBMCs.

[0400] dTp control (P, 5e6): Cryopreserved PBMCs were thawed and allowed to rest for 1 hour. Resting cells were harvested, spun down, and resuspended in Nucleofector buffer containing dTp control and plasmid TA (encoding SB11 transposase, as described in Example 1) at a final transposon:transposase ratio of 3:1 (Table 17). "(P, 5e6)" represents the total number of 5x10 injected 6 Refers to PBMC-derived cells.

[0401] Plasmid A (P, 5e6): Cryopreserved PBMCs were thawed and rested for 1 hour. Resting cells were harvested and resuspended in Nucleofector buffer containing Plasmid A and Plasmid TA at a final transposon:transposase ratio of 3:1 (Table 17). Similar to the dTp control, "(P, 5e6)" was used to measure the 5x10 6 Refers to PBMC-derived cells.

[0402] Plasmid A (T, 1e6) and Plasmid A (T, 0.5e6): Cryopreserved CD3 enriched cells were thawed and processed as described above for mock CD3. Resting cells were harvested and resuspended in Nucleofector buffer containing Plasmid A and Plasmid TA at a final transposon:transposase ratio of 3:1 (Table 17). "(T, 1e6)" represents the total number of 1x10 6 CD19CAR + CD3 + cells, and "(T, 0.5e6)" refers to the 0.5 x 10 6 CD19CAR + CD3 + Refers to cells.

[0403] For PBMC-derived RPM cells, immediately after electrotransfer, the contents from each cuvette were resuspended and transferred to R10 medium and allowed to rest for 1-2 hours in a 37°C / 5% CO2 incubator. A full medium change was then performed with R10 medium and the cells were placed in a 37°C / 5% CO2 incubator overnight. Within 24 hours after electrotransfer, cells were harvested from the cultures and sampled by flow cytometry to determine cell surface expression of CD19CAR, mbIL15, and HER1t, as well as other T cell markers, e.g., to characterize T cell memory subsets. To formulate for injection into mice, the desired cell number of each test article was resuspended in Plasmalyte A to achieve an injection volume of 300 μL per mouse.

[0404] For T cell derived RPM cells, immediately after electrotransfer, the contents from each cuvette were resuspended and transferred to R10 medium containing DNase for 1-2 hours of incubation in a 37°C / 5% CO2 incubator. A full medium change was then performed with R10 medium and the cells were placed in a 37°C / 5% CO2 incubator overnight. Within 24 hours after electrotransfer, cells were harvested from the cultures and sampled by flow cytometry to determine cell surface expression of CD19CAR, mbIL15, and HER1t, as well as other T cell markers, e.g., to characterize T cell memory subsets. Additionally, dead cells and debris were removed from the harvested cells, and the cells were enriched for viable cells. To formulate for injection into mice, the desired cell number of each test article was resuspended in Plasmalyte A to achieve an injection volume of 300 μL per mouse.

[0405] [Table 17]

[0406] 6.4.1.4 Animals Approximately 8-week-old female NOD / SCID / gamma mice (NOD.Cg-Prkdc scid Il2 rgtm1Wjl / SzJ, NSG) were purchased from Jackson Laboratory (Bar Harbor, ME). NSG mice lack both B and T lymphocytes and NK cells (e.g., as described in Ali et al., PLoS ONE. 2012; 7(8): e44219, the contents of which are incorporated herein by reference in their entireties). This line has ALL with excellent engraftment of human hematopoietic cells and the ability to detect blasts in peripheral blood (e.g., as described in Agliano et al., Int J Cancer. 2008; 123: 2222-2227, and Santos et al., Nat Med. 2009; 15(3): 338-344, the contents of each of which are incorporated herein by reference in their entireties). Test articles were manufactured and studies were performed at MDACC in compliance with its Institutional Animal Care and Use Committee (IACUC) and Guidelines for the Care and Use of Laboratory Animals (Eighth Edition, NRC, 2011, published by the National Academy Press, the contents of which are incorporated herein by reference in their entirety) and the Public Health Service Policy on Humane Care and Use of Laboratory Animals, Office of Laboratory Animal Welfare, Department of Health and Human Services (OLAW / NIH, 2002, the contents of which are incorporated herein by reference in their entirety). In a previous report, 6-12 week old NSG mice were cultured for 10 days without host preconditioning. 7 have been shown to efficiently engraft with human PBMCs and consistently develop xGvHD with accelerated weight loss and significantly faster disease onset (median survival time (MST)=40 days) (e.g., as described in Ali et al., PLoS ONE. 2012;7(8):e44219, the contents of which are incorporated herein by reference in their entirety).

[0407] 6.4.1.5 Study Design On day 1, NSG mice were inoculated with 1.5 x 10 cells in 0.2 mL of sterile PBS via the tail vein. 4 Viable NALM-6 / fLUC cells were injected into each control group. On day 6, animals underwent bioluminescence imaging (BLI) to detect the presence of tumors. Based on these data, animals were stratified into treatment groups, all of which observed similar mean tumor flux signals. Animals received test article treatment on day 7, as shown in Table 18, and the total cell counts in control groups B and C were consistent with the total cell counts of the corresponding genetically modified T cell treatment groups.

[0408] [Table 18]

[0409] 6.4.1.6 Animal handling and imaging methodology 6.4.1.6.1 Weight measurement Animals were weighed 2-3 times weekly for the duration of the study.

[0410] 6.4.1.6.2 In vivo BLI BLI is a sensitive, low-noise, non-invasive technique used to visualize, track, and monitor specific cellular activities in animals. Long-term monitoring of luminescent signals provides a quantitative assessment of tumor burden. Firefly luciferase (fLUC) from NALM6 was used as a bioluminescent reporter with D-luciferin provided as a substrate. BLI was performed using a Xenogen IVIS Spectrum In Vivo Imaging System (Xenogen, Caliper LifeSciences, Hopkinton, MA) on days 6, 14, 19, 22, 25, 28, 32, 35, 39, 42, 43, 46, 49, 53, 56, 60, and 62. Bioluminescence imaging datasets were acquired and quantified using Living Image software (v.4.5, Xenogen, Caliper LifeSciences, Hopkinton, MA). Ten minutes prior to imaging time, each mouse was administered a single subcutaneous (sq) injection of 214.5 μg D-luciferin (1.43 mg / mL working stock solution, Caliper) in 150 μL PBS. Animals were maintained on 2% isoflurane and placed in a biocontainment device (e.g., as described in Gade et al., Cancer Res. 2005;65(19):9080-9088, the contents of which are incorporated herein by reference in their entirety). Mice were imaged with exposure times determined by automatic exposure, except on day 6, when a 4-minute exposure acquisition was also performed. Abdominal images were acquired and quantified for each animal. Total flux values ​​were determined by drawing regions of interest (ROIs) of equivalent size across each mouse and presented in photons / s (p / s) (e.g., as described in Gade et al., Cancer Res. 2005;65(19):9080-9088, and Cooke et al., Blood. 1996;8(8):3230-3239, the contents of each of which are incorporated by reference in their entireties)."Background" BLI, which defines tumor-free mice (i.e., flux ≤ 2x background), is established using NSG mice injected with luciferin but without NALM-6 (and therefore no fLUC activity) with abdominal images captured.

[0411] 6.4.1.6.3 Blood collection Terminal bleeding was collected by retro-orbital bleeding and collected in sodium heparin-coated tubes. + The presence of T cells and tumor was determined by flow cytometry. Whenever possible, blood was collected from moribund animals. Samples were incubated in ACK lysis buffer (Thermo-Fisher) to lyse red blood cells, resuspended in PBS and 2% FBS, and kept at 4°C until immunostaining was performed (typically within 4 hours of tissue collection) to evaluate the presence of CD19CAR, mbIL15, and HER1t on T cells by flow cytometry.

[0412] 6.4.1.6.4 Clinical Observations and Endpoints Hydragel was placed in the cages of animals that appeared sick to aid in recovery. Mice were monitored daily for any signs of pain or other discomfort due to the treatment. Any signs of animal illness were recorded. Animals experiencing the following signs were humanely euthanized by cervical dislocation after informing and obtaining consent from the PI, following IACUC protocol: 1) failure to eat or drink for 24-48 hours resulting in wasting or dehydration; 2) consistent or rapid weight loss reaching 20% ​​at any time point or a 15% loss sustained for 72 hours compared to the pre-treatment weight of the mouse or age-matched vehicle-treated controls; 3) persistent hypothermia; 4) bloody or mucopurulent discharge from any orifice; 5) labored breathing especially if accompanied by nasal discharge and / or cyanosis; 6) enlarged lymph nodes or spleen; 7) hind limb paralysis or weakness; 8) significant abdominal distension or ascites burden exceeding 10% of the body weight of age-matched controls; 9) urinary incontinence or diarrhea for 48 hours; 10) lack of response to stimuli.

[0413] 6.4.1.6.4.1 Bioanalytical Assays Peripheral blood (PB), spleen, and BM samples were immunophenotyped and assessed by flow cytometry for the presence of NALM-6 / fLUC tumor cells and gene-modified T cells.

[0414] 6.4.1.6.4.2 Flow cytometry max 2×10 6Cells were stained with human-specific (unless otherwise stated) fluorochrome-conjugated antibodies. Staining of cell surface markers on samples and corresponding controls was first performed with an Fc receptor blocking step and background staining was reduced by incubation with 50% mouse serum (Jackson ImmunoResearch, PA) in FACS buffer (PBS, 2% FBS, 0.1% sodium azide) for 10 minutes at 4°C. Immunostaining was performed by adding 100 μL of antibody master mix of the antibody combinations listed in Table 19 diluted in Brilliant Stain Buffer (BD Biosciences). Briefly, CD19CAR expression was detected using an anti-idiotypic antibody (clone number 136.20.1) conjugated with Alexa Fluor (cryopreserved) (AF) 488 specific to the anti-CD19 portion of CD19CAR (e.g., as described in Jena et al., PLoS. 2013; 8(3): e57838, the contents of which are incorporated herein by reference in their entirety). The CD19CAR anti-idiotypic antibody was conjugated to the AF-488 fluorophore by Invitrogen / Thermo Fisher Scientific (Waltham, MA). HER1t molecules were detected using a fluorescently conjugated cetuximab antibody. The fluorescently conjugated cetuximab reagent was commercially purchased Erbitux conjugated to AF-647 by Invitrogen / Thermo Fisher Scientific. Fluorescently conjugated antibodies included CD8 (clone RPA-T8), CD3 (clone SK7), CD45RO (UCHL1), IL-15 (34559), CD45 (clone HI30), CCR7 (clone G043H7), CD19CAR idiotype (clone 136.20.1), and mouse CD45.1 (clone A20) (Table 19).

[0415] [Table 19]

[0416] Master mixes containing the antibody combinations in Table 19 were added sequentially (CD19CAR, mbIL15, followed by the remaining antibody cocktail) and incubated for up to 30 minutes at 4° C. between each addition. Cells were washed with FACS buffer and then incubated with fixable viability stain-620 viability dye (1:1000 in PBS, BD Biosciences) for 10 minutes at 4° C., followed by washing with FACS buffer. Data was acquired using an LSR Fortessa (BD Biosciences) with FACSDiva software (v.8.0.1, BD Biosciences) and analyzed with FlowJo software (version 10.4.2, TreeStar, Ashland, OR).

[0417] 6.4.1.6.5 Statistical analysis Statistical tests are described with each statistic reported. Post-hoc analyses were performed to compare differences between treatment groups and are reported with each statistical result. Errors are reported as standard deviations (SD). Statistical analyses were performed using GraphPad Prism (version 8) software. P<0.05 was considered statistically significant. Specific handling of total flux values ​​for statistical analysis involved log-transforming flux values ​​to address variance heterogeneity prior to significance testing.

[0418] 6.4.2 Generation and Evaluation of RPM T Cells Co-Expressing CD19CAR, mbIL15, and HER1t In Vivo 6.4.2.1 Genetic modification of T cells using the SB system to produce RPM CD19CAR-mbIL15-HER1t T cells Cell Process Generation of RPM T cells from PBMCs On day 1, a total of 3.68 x 10 cells were rested for 1 h and electroporated. 9 Starting with 1.12 × 10 PBMCs 9PBMCs / group were used to produce PBMC-derived test articles dTp control (P, 5e6) and Plasmid A (P, 5e6) as described in Table 17. On day 2 (approximately 18 hours after electrotransfer), 1.25 x 10 8 ~1.29×10 8 Viable cells were harvested.

[0419] T cell derived RPM For the T cell test arm, 3.00 × 10 9 Thaw enriched T cells, 1.70 x 10 9 Cells were allowed to recover after overnight rest. 1.26 × 10 9 cells were used for electrotransfer to produce plasmid A(T, 1e6) and plasmid A(T, 0.5e6). On day 3 (approximately 18 hours after electrotransfer), 4.23 × 10 8 Viable cells were harvested.

[0420] Approximately 18 hours after electrotransfer, T cells were cultured as singlets / live cells / CD3 + Transgene expression was assessed by flow cytometry as gated on events (Figures 22A-22C). Low transgene expression was detected with test articles from PBMCs, so mice were dosed at 1x10 6 CARs + Not cells, but a total of 5 × 10 6 The total viable cells were set at 10.

[0421] The remaining PBMC-derived test articles were then expanded ex vivo by three rounds of recurrent stimulation into activated proliferating cells (AaPCs) and supplemented with IL-21 (30 ng / mL) to confirm gene transfer. These expanded cells were assessed for the expected antigen-specific outgrowth of transgene-positive T cells. Less than 1% CAR+ and less than 1% mbIL15 were detected 18 hours after electroporation. + Despite the low HER1t expression and <4%, these RPM T cells showed visible and high transgene expression after numerical expansion (Figures 23A-23C). + CAR+ Events were 86% and 98% for dTp control (P, 5e6) and Plasmid A (P, 5e6) RPM T cells, respectively. dTp control (P, 5e6) cells showed population heterogeneity, consistent with previous examples. As shown in Figure 23B, the following percentages of CD19CAR / Her1t phenotype were observed: CD19CAR + HER1t + (50%), CD19CAR + HER1t 陰性 (27%), CD19CAR 陰性 HER1t+ (7%) and CD19CAR 陰性 HER1t 陰性 (16%). Similarly, as shown in FIG. 23C, the following percentages of the HER1t / mbIL15 phenotype were observed: HER1t + mbIL15 陰性 (49%), HER1t + mbIL15 + (7%), HER1t 陰性 mbIL15 + (<1%), and HER1t 陰性 mbIL15 陰性 (44%).

[0422] In contrast, as shown in Figure 23B, uniform CAR and HER1t co-expression was observed in plasmid A (P, 5e6) cells. CAR + HER1t + (94%), CAR + HER1t 陰性 (3%), C.A.R. 陰性 HER1t + (<1%), and CAR 陰性 HER1t 陰性 (2%). Similarly, co-expression of HER1t and mbIL15 was improved, as shown in Figure 23C. + mbIL15 陰性 (69%), HER1t + mbIL15 + (26%), HER1t 陰性 mbIL15 + (<1%), and HER1t 陰性 mbIL15陰性 (5%).

[0423] 6.4.2.2 Antitumor Effect of RPM CD19CAR-mbIL15-HER1t T Cells The antitumor efficacy of RPM CD19CAR-mbIL15-HER1t T cells was investigated in a NALM-6 mouse xenograft model. The study design is shown in Table 18, and the tumor burden results are shown in Figures 24A-24G. Notably, all mice bearing untreated tumors developed disease by day 35 (Figure 24A). Mice receiving mock PBMC developed disease by day 35, except for one mouse that died on day 7 due to injection complications and one mouse that reached day 46 (Figure 24B). In the mock CD3 treatment group, three of five mice developed disease between days 39 and 53 (tumor flux >1x10 9 p / s). Two mice showed suspected xGvHD (tumor flux <6×10), an expected outcome in the human lymphocyte-engrafted NSG model, on days 39 and 49. 7 p / s), one of the mice was moribund with a flux twice the background (<1.2 × 10 6 p / s) (Figure 24C). Mice treated with dTp control (P, 5e6) generally became moribund between days 35 and 62, with 2 out of 10 mice reaching a high disease burden (>5x10 9 p / s) and therefore likely disease-related mortality. The remaining mice showed stable disease or low tumor burden (xGvHD-related mortality), with 63% of them below or close to the threshold of 2x background flux (Figure 24D). Mice treated with plasmid A (P, 5e6) showed mortality between days 35 and 60, with one mouse having a high tumor burden and the remaining 8 mice having a low tumor burden (<7x10 7p / s) and likely xGvHD-related deaths, 75% of which were below or close to the two-fold background flux threshold (Figure 24E). Mice treated with plasmid A(T, 1e6) survived from day 35 to day 52, with 9 of 10 mice exhibiting low tumor burden (<5×10 7 p / s) and showed clear tumor signal decline in the days prior to endpoint, thus indicating xGvHD-related morbidity. Of the nine mice with rapidly declining tumors, four mice (44%) showed tumor signal below the two-fold background flux threshold (Figure 24F). Mice treated with plasmid A(T, 0.5e6) survived between days 32 and 60, with four of four mice showing low tumor burden (<8×10) at endpoint. 7 p / s) and therefore likely xGvHD-associated morbidity, 50% of them were close to the 2x background flux threshold (Figure 24G). In summary, all RPM CD19CAR-mbIL15-HER1t T cell test articles showed significant anti-tumor activity compared to untreated controls (<0.0006 in all groups, n=4-10, one-way ANOVA, Dunnett post-hoc test), and all showed significantly lower tumor burden compared to mock control cells, except for Plasmid A (T, 0.5e6) treatment, which did not achieve statistical significance with the group size tested (Figure 25). The kinetics of the anti-tumor response is consistent with previous studies and is typically observed to begin 20 days after tumor injection, thus approximately 2 weeks after T cell transfer.

[0424] Overall, the results suggest that established CD19 + Clearly demonstrating a potent antitumor response by RPM CD19CAR-mbIL15-HER1t T cells in the NALM-6 xenograft model.

[0425] 6.4.2.3 Overall Survival and Disease-Free Survival in Animals Treated with RPM CD19CAR-mbIL15-HER1t T Cells Administration of any of the RPM CD19CAR-mbIL15-HER1t T cell test articles (dTp control (P, 5e6), Plasmid A (P, 5e6), Plasmid A (T, 1e6), or Plasmid A (T, 0.5e6), corresponding to animal groups D-G, respectively) significantly enhanced OS in mice when compared to the tumor-only control group (P=0.0002, P=0.0004, P<0.0002, and P=0.0098 for groups D-G, respectively, n=4-10, log rank, Mantel-Cox, Figures 26A-26C). Tumor-only mice were moribund from disease at 5x10 9 p / s, indicating low tumor burden (total flux <1 × 10) likely due to xGvHD rather than disease progression in mice. 8 Mortality was observed in mice with p / s.

[0426] Induction of xGvHD is an expected process in NSG models engrafted with human lymphocytes (e.g., as described in Ali et al., PLoS ONE. 2012;7(8):e44219, the contents of which are incorporated by reference in their entirety). Taking that factor into account, xGvHD-free survival was calculated, whereby total flux <1×10 8 Animals with p / s were censored. In this analysis, survival was increased for all of the RPM CD19CAR-mbIL15-HER1t T cell test articles compared to the tumor-only control group (P=0.0002, P<0.0001, P<0.0001, and P=0.0018 for groups D-G, respectively, n=4-10, log rank, Mantel-Cox, Figures 27A-27C).

[0427] In summary, these results demonstrate that the tested RPM CD19CAR-mbIL15-HER1t T cells derived from both PBMCs and T cell enriched products conferred a significant increase in OS compared to tumor-only controls.

[0428] 6.4.2.4 Determination of xGvHD and Lack of Toxicity of RPM CD19CAR-mbIL15-HER1t T Cells in Mice No changes in body weight associated with the RPM CD19-mbIL15-CAR-T cell test article were observed prior to possible induction of the XGvHD process (i.e., within 1 week after T cell adoptive transfer), whereas Mock-PBMC and Mock-CD3 treatments showed weight loss during this period. Furthermore, over the course of the experiment, Mock-PBMC and Mock-CD3 treatments caused progressive weight loss in mice (i.e., the linear regression slope was negative and significantly different from 0; R 2 = 0.14 and R 2 = 0.44, slopes -0.06 and -0.11, P = 0.0123 and P < 0.001). No significant decrease in mouse body weight was observed in groups D to G during the experimental period (i.e., the linear regression slopes were significantly different from positive ± 0, R 2 <0.05, slope >0.03, P>0.02 for groups D-G. This suggests that groups B and C experienced xGvHD effects throughout the majority of the study, while groups D-G experienced a more sudden onset of morbidity just prior to becoming moribund. Tumor-only mice showed weight gain until they became moribund due to tumor burden.

[0429] In summary, intravenous administration of RPM CD19CAR-mbIL15-HER1t T cells in NALM-6 bearing mice (Groups D-G) was well tolerated. No toxicity was observed around the time of administration of the RPM test article (Groups D-G), and weight changes around the time of euthanasia were likely attributable to xGvHD.

[0430] 6.4.2.5 Persistence, Localization, and Memory Phenotype of RPM CD19CAR-mbIL15-HER1t T Cells To evaluate the persistence, localization, and memory phenotype of RPM CD19CAR-mbIL15-HER1t T cells, flow cytometry analysis was performed on peripheral blood (PB), bone marrow (BM), and spleen isolated from mice. Samples were obtained when mice became moribund or at the end of the study (study days 32-62). T cell engraftment was observed in all T cell treated mice (Mock PBMC, Mock CD3, dTp control (P, 5e6), Plasmid A (P, 5e6), Plasmid A (T, 1e6), and Plasmid A (T, 0.5e6, groups B-G, Figures 28A-28C, respectively). Engrafted CD3 + Among the cells, CAR + T cells were observed to persist at significant levels in the PB, BM, and spleen (Figure 29A) and in the PB (Figure 29B) of mice treated with RPM CD19CAR-mbIL15-HER1t T cells (groups D-G) ranging from 0% to 52%, 2% to 100%, 8% to 46%, and 15% to 74%, respectively, with no clear CD19CAR T cells in the mock PBMC and mock CD3 treated groups. + No clusters were detected. CAR + Similar frequencies of T cells were observed in the BM and spleen (Figures 29C-29D).

[0431] The primary goal of introducing tricistronic plasmid A genetic modifications in T cells was to reduce the heterogeneity of the transgene population. This was observed in samples evaluated for co-expression of CD19CAR and HER1t from cells isolated from PB. Plasmid A test article significantly increased the expression of CAR compared to the dTp control (P, 5e6). + HER1t +The results showed improved uniformity of expression of T cells (Figure 30). The detected co-expression with HER1t and mbIL15, and thus expression of mbIL15, was more variable (Figure 31) and was likely influenced by the circulating dynamics of mbIL15, possibly due to a mechanism for responding to cells internalizing IL-15 bound to IL-15Rα, which is cleaved from the presenting cells (see, e.g., Tamzalit et al., Proc Natl Acad Sci US A. 2014;111(23):8565-8570, the contents of which are incorporated herein by reference in their entirety). Nevertheless, there were cases of high co-expression with HER1t and mbIL15 (e.g., in the plasmid A(T, 0.5e6) sample). Importantly, the HER1t present under in vivo conditions was not significantly different from the IL-15Rα-dependent IL-15 expression. 陰性 mbIL15 + There was no significant clumping of cells (Figure 31).

[0432] CD19CAR memory phenotype persists in the PB of dying mice + CD3 + T cells were evaluated. The T cell memory subset is defined as: CD45RO + CCR7 + :Central Memory (T CM );CD45RO 陰性 CCR7 + :Naive / Stem Cell Memory (T N / SCM );CD45RO + CCR7 陰性 :Effector Memory (T EM ), and CD45RO 陰性 CCR7 陰性 :Effector T(T Eff Furthermore, T cell differentiation (low to high) is regulated by CD45RO 陰性 CD27 + , CD45RO + CD27 + , CD45RO + CD27 陰性 , and CD45RO 陰性 CD27 陰性The CD19CAR that was found to persist may be expressed as: + CD3 + T cells are primarily EM (Figure 32A), and when CD45RO and CCR7 were used as classification criteria, the means ranged from 59% to 70% in the RPM test articles (Groups D-G) (Figure 33A). However, predominant CD27 expression was observed in Groups D-G (Figure 32B), and the means were + CD27 + CD19CAR + CD3 + 33%-51% of T cells are poorly differentiated CD45RO 陰性 CD27 + CAR + CD3 + In T cells, expression ranged from 14% to 31% (Figure 33B). CD27 expression is indicative of a non-terminally differentiated, less differentiated memory phenotype (see, e.g., Larbi and Fulop, Cytometry A. 2014;85(1):25-35, the contents of which are incorporated herein by reference in their entirety).

[0433] Overall, these data demonstrate that all of the RPM CD19CAR-mbIL15-HER1t T cell test articles evaluated inhibited CD27-expressing T EM As such, these compounds persisted primarily in vivo until the endpoint.

[0434] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0435] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0436] Other embodiments are within the scope of the following claims.

Claims

1. A recombinant vector comprising a polycistronic expression cassette, wherein the expression cassette is arranged in a 5' to 3' direction as follows: (a) a first polynucleotide encoding a chimeric antigen receptor (CAR) comprising (i) an extracellular antigen-binding domain that specifically binds to CD19, (ii) a transmembrane domain, and (iii) a cytoplasmic domain; (b) a second polynucleotide sequence comprising an F2A element; and (c) a third polynucleotide encoding a fusion protein comprising (i) IL-15, or a functional fragment or functional variant thereof, and (ii) IL-15Rα, or a functional fragment or functional variant thereof; (d) a fourth polynucleotide sequence comprising a T2A element; and (e) a fifth polynucleotide encoding a marker protein; and A recombinant vector comprising a transcriptional regulatory element operably linked to a polynucleotide comprising:

2. A recombinant vector as described in claim 1, wherein the transcriptional regulatory element is a promoter.

3. The recombinant vector of claim 2, wherein the promoter is a human elongation factor 1-alpha (hEF-1α) hybrid promoter.

4. The recombinant vector of claim 1, further comprising a left inverted terminal repeat (ITR) and a right ITR adjacent to the polycistronic expression cassette.

5. The recombinant vector described in claim 4, wherein the ITR is selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, TcBuster transposon, and Tol2 transposon.

6. The recombinant vector described in claim 1, wherein the extracellular domain of the CAR comprises a single-chain variable fragment (scFv).

7. A recombinant vector as described in claim 6, wherein the scFv comprises a VH and a VL that bind to CD19.

8. The recombinant vector described in claim 7, wherein VH and VL are derived from monoclonal antibody FMC63.

9. The antigen-binding domain comprises: (a) a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3; (b) a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; The recombinant vector of claim 1 , comprising:

10. A recombinant vector as described in claim 9, wherein VH comprises a sequence having at least 95% identity to sequence number 2 and VL comprises a sequence having at least 95% identity to sequence number 1.

11. The recombinant vector described in claim 10, wherein the antigen-binding domain comprises a sequence having at least 95% identity to sequence number 12.

12. The recombinant vector described in claim 1, wherein the CAR further comprises a hinge region located between the antigen-binding domain and the transmembrane domain.

13. A recombinant vector as described in claim 12, wherein the hinge region comprises an amino acid sequence having at least 95% identity to sequence number 37.

14. A recombinant vector as described in claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

43.

15. A recombinant vector as described in claim 1, wherein the cytoplasmic domain comprises the primary signaling domain of human CD3ζ, or a functional fragment or functional variant thereof.

16. The recombinant vector of claim 7, wherein the CAR further comprises a costimulatory domain, or a functional fragment or variant thereof, derived from CD28, 4-1BB, OX40, CD2, CD7, CD27, CD30, CD40, CD5, ICAM-1, LFA-1, B7-H3, or ICOS.

17. The recombinant vector described in claim 15, wherein the primary signaling domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

61.

18. The recombinant vector described in claim 1, wherein the CAR comprises an amino acid sequence having at least 90% identity to sequence number 74.

19. The fusion protein, (a) IL-15 or a functional fragment or functional variant thereof, and (b) IL-15Rα or a functional fragment or functional variant thereof The recombinant vector of claim 1, further comprising a peptide linker linking 20. The recombinant vector of claim 19, wherein the peptide linker comprises the sequence of SEQ ID NO: 125 or a sequence that differs from SEQ ID NO: 125 by up to five amino acid substitutions.

21. A recombinant vector as described in claim 1, wherein the fusion protein comprises a polypeptide having at least 95% identity to SEQ ID NO: 123 and a polypeptide having at least 95% identity to SEQ ID NO:

124.

22. A recombinant vector as described in claim 1, wherein the fusion protein has at least 95% identity to sequence number 121.

23. The recombinant vector of claim 1, wherein the marker protein comprises a truncated EGFR1 or a functional fragment or functional variant thereof.

24. The marker protein (a) HER1 domain III or a functional fragment or functional variant thereof, and (b) the N-terminal portion of HER1 domain IV or a functional fragment or functional variant thereof The recombinant vector of claim 1 , comprising:

25. The marker protein (a) a polypeptide having at least 95% identity to SEQ ID NO: 98, and (b) a polypeptide having the sequence of SEQ ID NO: 100 or a sequence that differs from SEQ ID NO: 100 by up to three amino acid substitutions. The recombinant vector of claim 1 , comprising:

26. The recombinant vector described in claim 23, wherein the marker protein further comprises a CD28 transmembrane domain, or a functional fragment or functional variant thereof.

27. The recombinant vector of claim 26, wherein the CD28 transmembrane domain or a functional fragment or functional variant thereof comprises the sequence of SEQ ID NO: 101 or a sequence that differs from SEQ ID NO: 101 by up to three amino acid substitutions.

28. The recombinant vector described in claim 1, wherein the marker protein comprises a sequence having at least 95% identity to sequence number 97.

29. The recombinant vector described in claim 1, wherein the vector is a plasmid.

30. The recombinant vector described in claim 1, wherein the vector is a viral vector.

31. The recombinant vector described in claim 1, wherein the viral vector is an adenoviral vector.

32. The recombinant vector described in claim 1, wherein the vector is a non-viral vector.

33. A host cell comprising a recombinant vector described in any one of claims 1 to 32.

34. The host cell described in claim 33, wherein the host cell is an immune effector cell.

35. The host cell described in claim 33, wherein the host cell is a T cell.

36. A method for producing a genetically modified cell, comprising transducing or transfecting a host cell with a recombinant vector according to any one of claims 1 to 32.

37. A composition comprising the host cell of claim 33 for use in treating a disease or disorder.

38. The composition described in claim 37, wherein the disease or disorder is characterized by overexpression of CD19 on the cell surface compared to expression of CD19 in cells not affected by the disease or disorder.

39. The disease or disorder is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), Ph-like acute lymphoblastic leukemia (Ph-like ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), chronic lymphocytic lymphoma, small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma 38. The composition of claim 37, wherein the disease is selected from the group consisting of primary mediastinal large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom's macroglobulinemia.

40. The composition described in claim 37, wherein the disease or disorder is B-ALL or MCL.

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