Recombinant vectors containing polycistronic expression cassettes and methods of use thereof

Abstract

Provided herein is a vector comprising a polycistronic expression cassette, the polycistronic expression cassette comprising a polynucleotide encoding a TCR alpha chain, a polynucleotide encoding a TCR beta chain, and a polynucleotide encoding a cytokine, wherein the polynucleotides are separated by a polynucleotide sequence comprising a 2A element.

Description

[Technical field]

[0001] 1.Technical Field The present disclosure relates to polycistronic vectors containing at least three cistrons and methods of using them. [Background technology]

[0002] 2. Background technology Co-expression of multiple genes in each cell of a population is important for a wide variety of biomedical applications. 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.

[0003] 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

[0004] 3. Overview of the Invention The present disclosure provides a recombinant vector comprising a polycistronic expression cassette comprising a transcriptional regulatory element operably linked to a polycistronic polynucleotide.

[0005] Provided herein is a recombinant vector comprising a polycistronic expression cassette, the polycistronic expression cassette comprising a transcriptional regulatory element operably linked to a polycistronic polynucleotide, the polycistronic polynucleotide comprising: a first polynucleotide sequence encoding a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region; a second polynucleotide sequence comprising a first 2A element; a third polynucleotide sequence encoding a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ) region; a fourth polynucleotide sequence comprising a second 2A element; and a fifth 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.

[0006] In some embodiments, either or both of the first 2A element and the second 2A element are independently a P2A element, a T2A element, an F2A element, or an E2A element.

[0007] In some embodiments, the first 2A element is a P2A element.

[0008] In some embodiments, the P2A element comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 18 or 20, or the amino acid sequence of SEQ ID NO: 18 or 20 containing one, two, or three amino acid modifications.

[0009] In some embodiments, the P2A element comprises 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: 19 or 21.

[0010] In some embodiments, the second 2A element is a T2A element.

[0011] In some embodiments, the T2A element comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 22 or 24, or the amino acid sequence of SEQ ID NO: 22 or 24 containing 1, 2, or 3 amino acid modifications.

[0012] In some embodiments, the T2A element comprises 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:23 or 25.

[0013] In some embodiments, either or both of the second polynucleotide sequence and the fourth polynucleotide sequence independently encode a furin recognition site.

[0014] In some embodiments, the furin recognition site comprises the amino acid sequence of SEQ ID NO: 2 or 4, or the amino acid sequence of SEQ ID NO: 2 or 4 containing 1, 2, or 3 amino acid modifications.

[0015] In some embodiments, the furin recognition site is encoded by the polynucleotide sequence of SEQ ID NO: 3 or 5, or the polynucleotide sequence of SEQ ID NO: 3 or 5 containing 1, 2, or 3 nucleotide modifications.

[0016] In some embodiments, the second polynucleotide sequence comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:10, or the amino acid sequence of SEQ ID NO:10 containing one, two, or three amino acid modifications.

[0017] In some embodiments, the second polynucleotide sequence comprises 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:11.

[0018] In some embodiments, the fourth polynucleotide sequence comprises a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 containing one, two, or three amino acid modifications.

[0019] In some embodiments, the fourth polynucleotide sequence comprises 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:13.

[0020] In some embodiments, IL-15, or a functional fragment or variant thereof, comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:76.

[0021] In some embodiments, IL-15, or a functional fragment or variant thereof, 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:77.

[0022] In some embodiments, IL-15Rα, or a functional fragment or functional variant thereof, comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:78.

[0023] In some embodiments, IL-15Rα, or a functional fragment or variant thereof, 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:79.

[0024] In some embodiments, IL-15, or a functional fragment or variant thereof, is operably linked to IL-15Rα, or a functional fragment or variant thereof, via a peptide linker.

[0025] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 81, or the amino acid sequence of SEQ ID NO: 81 containing one, two, or three amino acid modifications.

[0026] In some embodiments, the peptide linker 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.

[0027] In some embodiments, the fusion protein is membrane bound.

[0028] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 70 or 73.

[0029] In some embodiments, the fusion protein 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:71 or 74.

[0030] In some embodiments, the Cα region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:40-49.

[0031] In some embodiments, the Cα region 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:55, 57 or 58.

[0032] In some embodiments, the Cβ region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50-54 or 60.

[0033] In some embodiments, the Cβ region 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:56 or 59.

[0034] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a first polynucleotide sequence, a second polynucleotide sequence, a third polynucleotide sequence, a fourth polynucleotide sequence, and a fifth polynucleotide sequence.

[0035] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:160, or the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:168, or the third polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a third combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:161.

[0036] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:230, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:231, or the third combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:232.

[0037] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:160, and the third polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a third combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:161.

[0038] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 180 or 210, and the third polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a third combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 181.

[0039] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:230 and the third combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:232.

[0040] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:250 or 270, and the third combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:252.

[0041] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a first polynucleotide sequence, a fourth polynucleotide sequence, a third polynucleotide sequence, a second polynucleotide sequence, and a fifth polynucleotide sequence.

[0042] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:162, or the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:166, or the third polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a sixth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:163.

[0043] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:233, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:234, or the sixth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:235.

[0044] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:162, and the third polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a sixth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:163.

[0045] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 182 or 212, and the third polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a sixth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 183.

[0046] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:233 and the sixth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:235.

[0047] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:253 or 273, and the sixth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:255.

[0048] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a first polynucleotide sequence, a second polynucleotide sequence, a fifth polynucleotide sequence, a fourth polynucleotide sequence, and a third polynucleotide sequence.

[0049] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:160; the first polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a seventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:169; the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:173; or the first polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, and the fourth polynucleotide sequence together comprise a ninth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:164.

[0050] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:230, the seventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:236, the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:237, or the ninth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:238.

[0051] In some embodiments, the first polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, and the fourth polynucleotide sequence together comprise a ninth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:164, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:50.

[0052] In some embodiments, the first polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, and the fourth polynucleotide sequence together comprise a ninth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 184 or 214, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 51.

[0053] In some embodiments, the ninth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:238 and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:59.

[0054] In some embodiments, the ninth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:258 or 278, and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:56.

[0055] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a first polynucleotide sequence, a fourth polynucleotide sequence, a fifth polynucleotide sequence, a second polynucleotide sequence, and a third polynucleotide sequence.

[0056] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:162; the first polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a tenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:167; the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:172; or the first polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, and the second polynucleotide sequence together comprise a twelfth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:165.

[0057] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:233, the tenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:239, the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:240, or the twelfth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:241.

[0058] In some embodiments, the first polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, and the second polynucleotide sequence together comprise a twelfth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:165, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:50.

[0059] In some embodiments, the first polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, and the second polynucleotide sequence together comprise a twelfth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 185 or 215, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 51.

[0060] In some embodiments, the twelfth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:241 and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:59.

[0061] In some embodiments, the twelfth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:261 or 281, and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:56.

[0062] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a third polynucleotide sequence, a second polynucleotide sequence, a first polynucleotide sequence, a fourth polynucleotide sequence, and a fifth polynucleotide sequence.

[0063] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:162, or the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:166, or the first polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a tenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:167.

[0064] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:233, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:234, or the tenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:239.

[0065] In some embodiments, the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:166, and the first polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a tenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:167.

[0066] In some embodiments, the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 186, and the first polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a tenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 187 or 217.

[0067] In some embodiments, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:234, and the tenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:239.

[0068] In some embodiments, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:254 and the tenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:259 or 279.

[0069] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a third polynucleotide sequence, a fourth polynucleotide sequence, a first polynucleotide sequence, a second polynucleotide sequence, and a fifth polynucleotide sequence.

[0070] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 160, or the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 168, or the first polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a seventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 169.

[0071] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:230, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:231, or the seventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:236.

[0072] In some embodiments, the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:168, and the first polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a seventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:169.

[0073] In some embodiments, the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 188, and the first polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a seventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 189 or 219.

[0074] In some embodiments, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:231 and the seventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:236.

[0075] In some embodiments, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:251 and the seventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:256 or 276.

[0076] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a third polynucleotide sequence, a second polynucleotide sequence, a fifth polynucleotide sequence, a fourth polynucleotide sequence, and a first polynucleotide sequence.

[0077] In some embodiments, the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:166; the third polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence together comprise a sixth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:163; the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:173; or the third polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, and the fourth polynucleotide sequence together comprise a thirteenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:170.

[0078] In some embodiments, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:234, the sixth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:235, the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:237, or the thirteenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:242.

[0079] In some embodiments, the third polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, and the fourth polynucleotide sequence together comprise a thirteenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:170, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:40.

[0080] In some embodiments, the third polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, and the fourth polynucleotide sequence together comprise a thirteenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 190, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 41 or 42.

[0081] In some embodiments, the thirteenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:242 and the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:57.

[0082] In some embodiments, the thirteenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:262 and the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:55 or 58.

[0083] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a third polynucleotide sequence, a fourth polynucleotide sequence, a fifth polynucleotide sequence, a second polynucleotide sequence, and a first polynucleotide sequence.

[0084] In some embodiments, the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:168; the third polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence together comprise a third combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:161; the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:172; or the third polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, and the second polynucleotide sequence together comprise a fourteenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:171.

[0085] In some embodiments, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:231, the third combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:232, the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:240, or the fourteenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:243.

[0086] In some embodiments, the third polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, and the second polynucleotide sequence together comprise a fourteenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:171, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:40.

[0087] In some embodiments, the third polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, and the second polynucleotide sequence together comprise a fourteenth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:191, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:41 or 42.

[0088] In some embodiments, the fourteenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:243 and the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:57.

[0089] In some embodiments, the fourteenth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:263 and the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:55 or 58.

[0090] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a fifth polynucleotide sequence, a second polynucleotide sequence, a first polynucleotide sequence, a fourth polynucleotide sequence, and a third polynucleotide sequence.

[0091] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 162, or the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 172.

[0092] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:233 or the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:240.

[0093] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 162, the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 172, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:50.

[0094] In some embodiments, the first polynucleotide sequence and the fourth polynucleotide sequence together comprise a fourth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 182 or 212, the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 222, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 51.

[0095] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:233, the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:240, and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:59.

[0096] In some embodiments, the fourth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 253 or 273, the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 240, and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 56.

[0097] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a fifth polynucleotide sequence, a fourth polynucleotide sequence, a first polynucleotide sequence, a second polynucleotide sequence, and a third polynucleotide sequence.

[0098] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 160, or the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 173.

[0099] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:230, or the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:237.

[0100] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 160, the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 173, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 50.

[0101] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence together comprise a first combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 180 or 210, the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 223, and the third polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 51.

[0102] In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 230, the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 237, and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 59. In some embodiments, the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 250 or 270, the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 237, and the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 56.

[0103] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a fifth polynucleotide sequence, a second polynucleotide sequence, a third polynucleotide sequence, a fourth polynucleotide sequence, and a first polynucleotide sequence.

[0104] In some embodiments, the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 168, or the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 172.

[0105] In some embodiments, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:231 or the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:240.

[0106] In some embodiments, the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 168, the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 172, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 40.

[0107] In some embodiments, the third polynucleotide sequence and the fourth polynucleotide sequence together comprise a second combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 188, the fifth polynucleotide sequence and the second polynucleotide sequence together comprise an eleventh combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 222, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 41 or 42.

[0108] In some embodiments, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:231, the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:240, and the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:57.

[0109] In some embodiments, the second combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 251, the eleventh combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 240, and the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 55 or 58.

[0110] In some embodiments, the polycistronic polynucleotide comprises, in 5' to 3' order, a fifth polynucleotide sequence, a fourth polynucleotide sequence, a third polynucleotide sequence, a second polynucleotide sequence, and a first polynucleotide sequence.

[0111] In some embodiments, the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:166, or the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:173.

[0112] In some embodiments, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:234, or the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:237.

[0113] In some embodiments, the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:166, the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:173, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:40.

[0114] In some embodiments, the third polynucleotide sequence and the second polynucleotide sequence together comprise a fifth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 186, the fifth polynucleotide sequence and the fourth polynucleotide sequence together comprise an eighth combined polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 223, and the first polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 41 or 42.

[0115] In some embodiments, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:234, the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:237, and the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:57.

[0116] In some embodiments, the fifth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:254, the eighth combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:237, and the first combined polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:55 or 58.

[0117] In some embodiments, the polycistronic polynucleotide further comprises a sixth polynucleotide sequence comprising a third 2A element, and a seventh polynucleotide sequence comprising a marker protein.

[0118] In some embodiments, the third 2A element is a P2A element, a T2A element, an F2A element, or an E2A element.

[0119] In some embodiments, the 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.

[0120] In some embodiments, domain III of HER1, or a functional fragment or functional variant thereof, comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:104.

[0121] In some embodiments, the N-terminal portion of domain IV of HER1 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:105.

[0122] In some embodiments, the N-terminal portion of domain IV of HER1 comprises amino acids 1-21 of SEQ ID NO:105.

[0123] In some embodiments, the N-terminal portion of domain IV of HER1 comprises the amino acid sequence of SEQ ID NO: 106, or the amino acid sequence of SEQ ID NO: 106 containing one, two, or three amino acid modifications.

[0124] In some embodiments, the transmembrane region of CD28 comprises the amino acid sequence of SEQ ID NO: 107, or the amino acid sequence of SEQ ID NO: 107 comprising one, two, or three amino acid modifications.

[0125] In some embodiments, the marker protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:100, 103, or 112.

[0126] In some embodiments, the Vα region comprises complementarity determining region 1α (CDR1α), CDR2α, and CDR3α comprising the amino acid sequences of SEQ ID NOs: 1001+10n, 1002+10n, and 1003+10n, respectively, where n is an integer from 0 to 79. In some embodiments, n=0.

[0127] In some embodiments, the Vβ region comprises CDR1β, CDR2β, and CDR3β that comprise the amino acid sequences of SEQ ID NOs: 2001+10n, 2002+10n, and 2003+10n, respectively, where n is an integer from 0 to 79. In some embodiments, n=0.

[0128] In some embodiments, the Vα region comprises CDR1α, CDR2α, and CDR3α from a Vα region comprising the amino acid sequence of SEQ ID NO: 1004+10n, 1005+10n, 1006+10n, or 1007+10n, where n is an integer from 0 to 79. In some embodiments, n=0.

[0129] In some embodiments, the Vβ region comprises CDR1β, CDR2β, and CDR3β from a Vβ region comprising the amino acid sequence of SEQ ID NO: 2004+10n, 2005+10n, 2006+10n, or 2007+10n, where n is an integer from 0 to 79. In some embodiments, n=0.

[0130] In some embodiments, the Vα region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1004+10n, 1005+10n, 1006+10n, or 1007+10n, where n is an integer from 0 to 79. In some embodiments, n=0.

[0131] In some embodiments, the Vβ region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2004+10n, 2005+10n, 2006+10n, or 2007+10n, where n is an integer from 0 to 79. In some embodiments, n=0.

[0132] In some embodiments, the Vα region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1004+10n, and the Vβ region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2004+10n, where n is any one of 0 to 100. 79, the Vα region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1005+10n, and the Vβ region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2005+10n, where n is an integer from 0 to 79. and n is an integer, wherein the Vα region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1006+10n, and the Vβ region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2006+10n, where n is an integer from 0 to 79. wherein the Vα region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1007+10n, and the Vβ region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2007+10n, and n is an integer from 0 to 79. In some embodiments, n=0.

[0133] In some embodiments, the TCR alpha chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1008+10n, where n is an integer from 0 to 79. In some embodiments, the TCR alpha chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1009+10n, where n is an integer from 0 to 79.

[0134] In some embodiments, the TCR alpha chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1010+10n, where n is an integer from 0 to 79.

[0135] In some embodiments, the TCR beta chain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2008+10n, where n is an integer from 0 to 79. In some embodiments, n=0.

[0136] In some embodiments, the TCR beta chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2009+10n, where n is an integer from 0 to 79.

[0137] In some embodiments, the TCR beta chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2010+10n, where n is an integer from 0 to 79.

[0138] In some embodiments, the transcriptional regulatory element comprises a promoter.

[0139] In some embodiments, the promoter is a human elongation factor 1-alpha (hEF-1α) hybrid promoter.

[0140] In some embodiments, the promoter comprises 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:150.

[0141] In some embodiments, the recombinant vector further comprises a polyA sequence at the 3' end of the polycistronic expression cassette.

[0142] In some embodiments, the polyA sequence comprises 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:151.

[0143] In some embodiments, the recombinant vector further comprises a left inverted terminal repeat (ITR) and a right ITR, wherein the left ITR and the right ITR flank the polycistronic expression cassette.

[0144] In some embodiments, the recombinant vector comprises, in 5' to 3' order, a left ITR, a transcriptional regulatory element, a first polynucleotide sequence, a second polynucleotide sequence, a third polynucleotide sequence, a fourth polynucleotide sequence, a fifth polynucleotide sequence, and a right ITR.

[0145] In some embodiments, the recombinant vector is a non-viral vector.

[0146] In some embodiments, the non-viral vector is a plasmid.

[0147] In some embodiments, the recombinant vector is a viral vector.

[0148] In some embodiments, the recombinant vector is a polynucleotide.

[0149] Also provided herein are polynucleotides that encode an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 163, 164, 165, 167, 169, 170, and 171.

[0150] Also provided herein are polynucleotides comprising a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 232, 235, 236, 238, 239, 241, 242, and 243.

[0151] Also provided herein are populations of cells comprising any of the recombinant vectors provided herein, or any of the polynucleotides provided herein.

[0152] In some embodiments, the recombinant vector or polynucleotide is integrated into the genome of the cell population.

[0153] In some embodiments, the cell is an immune effector cell.

[0154] In some embodiments, the 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.

[0155] In some embodiments, the immune effector cell is a T cell.

[0156] In some embodiments, the T cells are selected from the group consisting of naive T cells (CD4+ or CD8+), killer CD8+ T cells, cytotoxic CD4+ T cells, helper CD4+ T cells, CD4+ T cells corresponding to Th1, Th2, Th9, Th17, Th22, follicular helper (Tfh), regulatory (Treg) lineages, tumor infiltrating lymphocytes (TIL), and memory T cells (central memory, effector memory, stem cell memory, stem cell-like memory).

[0157] In some embodiments, the cell population comprises alpha / beta T cells, gamma / delta T cells, or natural killer T (NKT) cells.

[0158] In some embodiments, the cell population comprises CD4+ T cells, CD8+ T cells, or both CD4+ T cells and CD8+ T cells.

[0159] In some embodiments, the cells are ex vivo.

[0160] In some embodiments, the cells are human.

[0161] In some embodiments, the cell population is T cells that comprises greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% CD45RA+CD45RO-CD62L+CD95+ cells.

[0162] In some embodiments, the cell population is T cells that comprises greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% CD45RA+CD45RO+CD62L+CD95+ cells.

[0163] Also provided herein is a cell population comprising a polycistronic expression cassette comprising a first cistron comprising a 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 second cistron comprising a polynucleotide sequence encoding a TCR beta chain comprising a Vβ region and a Cβ region, and a third cistron comprising a polynucleotide sequence encoding a TCR alpha chain comprising a Vα region and a Cα region, wherein the cell population are T cells comprising more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% CD45RA+CD45RO-CD62L+CD95+ cells.

[0164] Also provided herein is a cell population comprising a multicistronic expression cassette comprising a first cistron comprising a 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 second cistron comprising a polynucleotide sequence encoding a TCR beta chain comprising a Vβ region and a Cβ region, and a third cistron comprising a polynucleotide sequence encoding a TCR alpha chain comprising a Vα region and a Cα region, wherein the cell population are T cells that comprise more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% CD45RA+CD45RO+CD62L+CD95+ cells.

[0165] Also provided herein is a method of producing a population of engineered cells, the method comprising introducing into a cell population any of the recombinant vectors provided herein and a DNA transposase, or a polynucleotide encoding the DNA transposase, and culturing the cell population under conditions in which the transposase integrates the polycistronic expression cassette into the genome of the cell population, thereby producing a population of engineered cells.

[0166] 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.

[0167] In some embodiments, the DNA transposon is a Sleeping Beauty transposon.

[0168] In some embodiments, the transposase is a Sleeping Beauty transposase.

[0169] In some embodiments, the Sleeping Beauty transposase is selected from the group consisting of SB11, SB10, SB100X, hSB110, and hSB81.

[0170] In some embodiments, the Sleeping Beauty transposase is SB11.

[0171] In some embodiments, SB11 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:300.

[0172] In some embodiments, SB11 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:301.

[0173] In some embodiments, the polynucleotide encoding the DNA transposase is a DNA vector or an RNA vector.

[0174] In some embodiments, the left ITR comprises a polynucleotide sequence 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:290 or 291, and the right ITR comprises a polynucleotide sequence 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:292, 293, or 294.

[0175] In some embodiments, the recombinant vector and the DNA transposase or a polynucleotide encoding the DNA transposase are introduced into the cell population using electroporation, sonication, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or mechanical deformation by passage through a microfluidic device, or a colloidal dispersion system.

[0176] In some embodiments, the recombinant vector and the DNA transposase or a polynucleotide encoding the DNA transposase are introduced into the cell population using electroporation.

[0177] In some embodiments, the method is completed within 30, 25, 20, 15, 14, 10, 7, 6, 5, 4, 3, 2, or 1 day.

[0178] In some embodiments, the method is completed in less than 30, 25, 20, 15, 14, 10, 7, 6, 5, 4, 3, 2, or 1 day.

[0179] In some embodiments, the cell population is cryopreserved and thawed prior to introduction of the recombinant vector and the DNA transposase or a polynucleotide encoding a DNA transposase.

[0180] In some embodiments, the cell population is rested prior to introduction of the recombinant vector and the DNA transposase or a polynucleotide encoding a DNA transposase.

[0181] In some embodiments, the cell population is not rested prior to introduction of the recombinant vector and the DNA transposase or a polynucleotide encoding a DNA transposase.

[0182] In some embodiments, the cell population comprises expanded human ex vivo cells.

[0183] In some embodiments, the cell population is not activated ex vivo.

[0184] In some embodiments, the cell population comprises T cells.

[0185] Also provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the cell populations provided herein, thereby treating the cancer.

[0186] In some embodiments, the cancer is selected from lung cancer, bile duct cancer, pancreatic cancer, colorectal cancer, gynecological cancer, and ovarian cancer.

[0187] Also provided herein is 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 any of the cell populations provided herein, thereby treating the autoimmune disease or disorder. [Brief description of the drawings]

[0188] 4. Brief description of the drawings The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure.

[0189] [Figure 1] A series of schematic diagrams of the structures of TCRα (A), TCRβ (B), and mbIL15 (15), shown from N-terminus (left) to C-terminus (right). [Figure 2A] FIG. 1 is a schematic diagram of the sequence of ORFs of the tricistronic cassettes APBT15, ATBP15, AP15TB, AT15PB, BPAT15, BTAP15, BP15TA, and BT15PA. [Figure 2B] 1 is a schematic diagram of the sequence of the ORFs of regulatory cassette 15, APB, and BPA. [Diagram 3] FIG. 1 is a schematic diagram showing double transposition and single transposition approaches using the Sleeping Beauty transposon / transposase system to generate T cells expressing TCRα / TCRβ and mbIL15. [Figure 4] FIG. 1 is a series of two-parameter flow plots showing transgene co-expression assessed after electroporation and overnight incubation for each of groups 1-14. [Figure 5A] FIG. 1 is a series of two-parameter flow plots showing representative TCR transgene expression in CD3+ cells after overnight incubation for each of groups 1-14. [Figure 5B] TCR expression data from three donors presented as %mTCR+ cells among CD3+ cells are provided. [Figure 6A] Expression of TCR and mbIL15 after the first expansion phase (day 13) is shown. Figure 6A provides representative TCR and mbIL15 expression data from each of groups 1-14. Figure 6B provides TCR expression data from three donors presented as %mTCR+ cells among CD3+ cells. Figure 6C provides TCR and mbIL15 co-expression data from three donors presented as %TCR+mbIL15+ cells among CD3+ cells. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7A]The total number of TCR+ and TCR+mbIL15+ cells after the first expansion phase (day 13) is shown. Figure 7A provides TCR expression data from three donors presented as the total number of mTCR+ T cells. Figure 7B provides the total number of TCR+mbIL15+ T cells from three donors. [Figure 7B] Same as above. [Figure 8A] For each of groups 1 to 14, cell viability after electroporation (day 1, FIG. 8A) and after the first expansion phase (day 13, FIG. 8B) is shown. [Figure 8B] Same as above. [Figure 9A] Figure 1 shows the specific induction of activation marker 4-1BB following overnight co-culture of translocated T cells from each of groups 1-14 after the first expansion phase (day 13) with wild-type or mutant neoantigen-pulsed T2 cells. Data are presented as % 4-1BB positive cells of CD8+ cells at increasing concentrations of neoantigen peptide. [Figure 9B] Same as above. [Figure 10] Shown are phosphorylated STAT5 levels in CD3+ T cells transferred from each of groups 1-14 after the first expansion phase (day 13). An isotype negative control and an IL-15 treated positive control were included for comparison (dTp = double transduced with separate mbIL15 and TCR vectors). [Figure 11] Apoptosis levels in translocated T cells from each of groups 2-14 are shown after expansion for 13 days followed by activation with CD3 / CD28 Dynabeads® (ThermoFisher) for 9 days. [Figure 12] FIG. 1 is a series of schematic diagrams showing the differences between the S and N versions of the TCR only and the mbIL15 TCR construct shown from N-terminus (left) to C-terminus (right). [Figure 13A] TCR expression on CD3+ cells is shown the day after electroporation (day 1), after the first expansion phase before enrichment (day 11 pre-enrichment), and (FIG. 13B) after the first expansion phase after enrichment (day 11 post-enrichment), and at the end of the second expansion phase (FIG. 13A). Data from four donors are presented. [Figure 13B]Same as above. [Figure 14A] Co-expression of TCR and mbIL15 on CD3+ T cells is shown the day after electroporation (day 1), after the first expansion phase before enrichment (day 11 pre-enrichment), and after the first expansion phase after enrichment (day 11 post-enrichment), and at the end of the second expansion phase (FIG. 14A). Data from four donors are presented. [Figure 14B] Same as above. [Figure 15] FIG. 1 is a series of two-parameter flow plots showing representative TCR and mbIL15 transgene expression in CD3+ cells after the second expansion phase. [Figure 16A] The proliferation rate of cells after the first expansion phase (FIG. 16A) and the second expansion phase (FIG. 16B) is shown. Data from four donors are presented. [Figure 16B] Same as above. [Figure 17A] Absolute numbers of TCR-expressing cells are shown in cultures after the first expansion phase (FIG. 17A) and after the second expansion phase (FIG. 17B). Data from four donors are presented. [Figure 17B] Same as above. [Figure 18] Figure 2 shows phosphorylated STAT5 levels after the second expansion phase (day 27) in CD3+ T cells transfected with different versions of a polycistronic plasmid encoding TCR001. Some contain non-cysteine ​​substituted TCR constant regions (N version) or are optionally further codon-optimized (NU version). Non-transfected (NT) = NT (group 2.1), BPA (group 2.2), BPA-N (group 2.3), AP15TB (group 2.4), AP15TB-N (group 2.5), AP15TB-NU (group 2.6), BP15TA (group 2.7), BP15TA-N (group 2.8), and BP15TA-NU (group 2.9). [Figure 19A]Functional data from translocated T cells co-cultured with neoantigen-pulsed dendritic cells are shown. Figure 19A shows the specific induction of activation marker 4-1BB after overnight co-culture of translocated T cells from each of groups 2.1-2.9 after the second expansion phase (day 27) with wild-type or mutant neoantigen peptide-pulsed dendritic cells. Data are presented as % 4-1BB positive cells of CD8+ cells at increasing concentrations of neoantigen peptide. Figure 19B shows interferon gamma (IFN-γ) secretion after overnight co-culture of translocated T cells from each of groups 2.1-2.9 after the second expansion phase (day 27) with wild-type or mutant neoantigen-pulsed dendritic cells. [Figure 19B] Same as above. [Figure 20A] Figure 20 shows TCR expression and cell survival after 4 weeks of long-term cytokine withdrawal (LTWD) incubation in translocated cells from each of groups 2.2-2.9. Figure 20A shows expression of mTCR detected in the CD3+ gated population with a mouse TCR beta antibody. Figure 20B shows cell survival as a percent of viable cells recovered compared to the initial input number of cells at the start of LTWD. [Figure 20B] Same as above. [Figure 21A] Specific induction of the activation marker 4-1BB following overnight co-culture of translocated T cells from each of groups 2.2 to 2.9 after 4 weeks of LTWD incubation with wild-type or mutant neoantigen (10 μg / ml) pulsed dendritic cells is shown. [Figure 21B] Same as above. [Figure 22A] IFN-γ secretion following overnight co-culture of translocated T cells from each of groups 2.2 to 2.9 after 4 weeks of LTWD incubation with wild-type or mutant neoantigen (10 μg / ml) pulsed dendritic cells is shown. [Figure 22B] Same as above. [Figure 23A] FIG. 23 is a series of pie graphs showing the mean frequencies of in vivo CD3+ T cell memory and effector subsets at day 11 post-expansion (FIG. 23A), day 22 post-expansion (FIG. 23B), and after 4 weeks of LTWD culture (FIG. 23C) in cells transduced with the plasmids tested (groups 2.2-2.9). [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 24] A series of two-parameter flow plots showing representative TCR and mbIL15 transgene co-expression in CD3+ cells after overnight incubation (day 1), after the first expansion phase (day 11, pre- and post-enrichment), and after the second expansion phase (day 22) for each of groups 3.2-3.4 expressing TCR001+ / - mbIL15 (BPA-N, AP15TB-NU, and BP15TA-NU). [Figure 25A] Changes in the TCR+ population during the first expansion phase (day 1 vs. day 11 prior to enrichment) are shown for cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30). Each graph presents data from a separate TCR, TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 26A] Changes in the TCR+ population during the second expansion phase (day 11 vs. day 22 post-enrichment) for cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30). Each graph presents data from a separate TCR, TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 27A] Changes in the TCR+ / mbIL15+ population during the first expansion phase (day 1 vs. day 11 prior to enrichment) are shown for cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30). Each graph presents data from a separate TCR, TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 27B] Same as above. [Figure 27C] Same as above. [Figure 28A]Figure 1 shows the TCR+ / mbIL15+ population change during the second expansion phase (day 11 vs. day 22 post-enrichment) for cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30). Each graph presents data from a separate TCR, TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 28B] Same as above. [Figure 28C] Same as above. [Figure 29A] Figure 1 shows the specific induction of activation marker 4-1BB after overnight co-culture of translocated T cells from each of groups 3.1-3.30 after the second expansion phase (day 27) with wild-type (WT) or mutant (Mut) neoantigen-pulsed dendritic cells. Data are presented as %4-1BB positive cells of total CD3+, CD4+, or CD8+ T cells at increasing concentrations of neoantigen peptide. NT = non-translocated, TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 29B] Same as above. [Figure 29C] Same as above. [Figure 29D] Same as above. [Figure 29E] Same as above. [Figure 29F] Same as above. [Figure 29G] Same as above. [Fig. 29H] Same as above. [Figure 29I] Same as above. [Figure 30A] Figure 1 shows IFN-γ secretion after overnight co-culture of translocated T cells from each of groups 3.1-3.30 after the second expansion phase (day 27) with wild-type (WT) or mutant (Mut) neoantigen-pulsed dendritic cells. Data are presented as IFN-γ levels (pg / mL) at increasing concentrations of neoantigen peptide. NT = non-translocated, TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 30B] Same as above. [Figure 30C] Same as above. [Figure 30D] Same as above. [Figure 30E] Same as above. [Figure 30F] Same as above. [Figure 30G] Same as above. [Figure 30H] Same as above. [Figure 30I] Same as above. [Diagram 31] Specific lysis of the negative control (Mut+HLA-) tumor cell line AU565 and the target tumor cell line TYK-nu (Mut+HLA+) by T cells expressing TCR001+ / -mbIL15. NT = non-rearranged, TCR001 only = BPA-N, TCR001 with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 32A] Specific lysis of tumor cell lines by T cells expressing (FIG. 32A) TCR022+ / -mbIL15 or (FIG. 32B) TCR075+ / -mbIL15. Tumor cell lines were transfected with the appropriate HLA expression plasmid, pulsed with either wild type (WT) or mutant (Mut) peptides and co-cultured with T cells. NT=non-rearranged, TCR only=BPA-N, TCR with mbIL15=AP15TB-NU or BP15TA-NU. [Figure 32B] Same as above. [Diagram 33] Figure 1 shows the TCR+ population of cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30) after long-term cytokine withdrawal (LTWD). TCR only = BPA-N, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 34A] Figure 1 shows cell survival of cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30) after long-term cytokine withdrawal (LTWD). BPA-N(IL2) = TCR cultured only with IL2, NT = non-transduced, BPA-N = TCR only, TCR with mbIL15 = AP15TB-NU or BP15TA-NU. [Figure 34B] Same as above. [Figure 34C] Same as above. [Figure 35A]Specific induction of activation marker 4-1BB after overnight co-culture of cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30) after long-term cytokine withdrawal (LTWD) with wild-type or mutant neoantigen-pulsed dendritic cells. Data are presented as %4-1BB+ of either CD3+, CD4+, or CD8+ T cells. BPA-N(IL2)=TCR cultured only with IL2, TCR with mbIL15=AP15TB-NU or BP15TA-NU. [Figure 35B] Same as above. [Figure 35C] Same as above. [Figure 35D] Same as above. [Figure 35E] Same as above. [Fig. 35F] Same as above. [Figure 35G] Same as above. [Fig. 35H] Same as above. [Fig. 35I] Same as above. [Figure 36A] IFN-γ secretion after overnight co-culture of cells transduced with various TCR+ / -mbIL15 (groups 3.1-3.30) after long-term cytokine withdrawal (LTWD) with wild-type or mutant neoantigen-pulsed dendritic cells. BPA-N(IL2)=TCR cultured only with IL2, TCR with mbIL15=AP15TB-NU or BP15TA-NU. [Figure 36B] Same as above. [Figure 36C] Same as above. [Figure 37A] Comparison of 4-1BB induction in cells transduced before and after LTWD culture of various TCR+mbIL15 (3.1-3.30 groups) following overnight co-culture with wild-type or mutant neoantigen-pulsed dendritic cells. Data are presented as %4-1BB+ of either CD3+, CD4+, or CD8+ T cells. [Figure 37B] Same as above. [Figure 37C] Same as above. [Figure 37D] Same as above. [Figure 37E] Same as above. [Figure 37F] Same as above. [Figure 37G] Same as above. [Fig. 37H] Same as above. [Fig. 37I] Same as above. [Figure 38] 1 is a series of representative pie graphs showing the mean frequencies of live CD3+ T cell memory and effector subsets at day 11 post-expansion for TCR001-transposed cells expressed with mbIL15 from either AP15TB-NU or BP15TA-NU. [Figure 39] 1 is a series of representative pie graphs showing the mean frequencies of live CD3+ T cell memory and effector subsets at day 22 post-expansion for TCR001-transposed cells expressed with BPA-N or with mbIL15 expressed with either AP15TB-NU or BP15TA-NU. [Figure 40A] FIG. 1 is a set of pie charts showing the mean frequencies of in vivo CD3+ T cell memory and effector subsets in cells transduced with the tested plasmids (groups 3.1 to 3.30) after 4 weeks of LTWD culture. [Figure 40B] Same as above. [Figure 40C] Same as above. [Figure 40D] Same as above. [Figure 40E] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0190] 5. MODE FOR CARRYING OUT THEINVENTION The present disclosure provides recombinant polycistronic nucleic acid vectors comprising at least three cistrons, where a first cistron encodes an alpha chain of an artificial T cell receptor (TCR), a second cistron encodes a beta chain of the artificial TCR, and a third cistron encodes a fusion protein comprising IL-15 and IL-15Ralpha (e.g., mbIL15), or a functional fragment or variant thereof. In some embodiments, the polycistronic nucleic acid further comprises a fourth cistron encoding a marker protein (e.g., HER1t). In some embodiments, the cistrons are separated by a polynucleotide sequence comprising a 2A element. Also provided are immune effector cells comprising these vectors, immune effector cells engineered ex vivo using the vectors to express three proteins encoded by the vectors, pharmaceutical compositions comprising these vectors or engineered immune effector cells produced using these vectors, and methods of treating a subject using these vectors or engineered immune effector cells produced using these vectors.

[0191] The polycistronic vectors described herein are particularly useful in methods for 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.

[0192] 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 dictates 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.

[0193] 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.

[0194] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and refer to molecules that contain CDRs or variable regions from an αβ T cell receptor. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs that contain variable regions of TCRs joined by flexible linkers, TCR chains linked by engineered disulfide bonds, single TCR variable domains, single peptide-MHC specific TCRs, multispecific TCRs (including bispecific TCRs), TCR fusions, TCRs containing costimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. In certain embodiments, the TCR is a full-length TCR that contains a full-length α chain and a full-length β chain. In certain embodiments, the TCR is a soluble TCR that lacks a transmembrane and / or cytoplasmic region. In certain embodiments, the TCR is a single chain TCR (scTCR) comprising Vα and Vβ linked by a peptide linker, such as a scTCR having a structure described in PCT Publication Nos. 2003 / 020763, 2004 / 033685, or 2011 / 044186, each of which is incorporated herein by reference in its entirety. In certain embodiments, the TCR comprises a transmembrane region. In certain embodiments, the TCR comprises a costimulatory signaling region.

[0195] As used herein, the term "full-length TCR" refers to a TCR comprising a dimer of a first and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In certain embodiments, a full-length TCR comprises one or two unmodified TCR chains, e.g., an unmodified αTCR chain or an unmodified βTCR chain. In certain embodiments, a full-length TCR comprises one or two modified TCR chains, such as a chimeric TCR chain and / or a TCR chain, that comprises one or more amino acid substitutions, insertions, or deletions compared to the unmodified TCR chain. In certain embodiments, a full-length TCR comprises a mature full-length TCR α chain and a mature full-length TCR β chain.

[0196] As used herein, the term "TCR variable region" refers to a portion of a mature TCR polypeptide chain (e.g., a TCR alpha or beta chain) that is not encoded by the TRAC gene for the TCR alpha chain, either the TRBC1 or TRBC2 genes for the TCR beta chain, or the TRDC gene for the TCR delta chain. In some embodiments, the TCR variable region of the TCR alpha chain encompasses all of the amino acids of the mature TCR alpha chain polypeptide encoded by the TRAV and / or TRAJ genes, and the TCR variable region of the TCR beta chain encompasses all of the amino acids of the mature TCR beta chain polypeptide encoded by the TRBV, TRBD, and / or TRBJ genes (see, e.g., Lefranc and Lefranc, (2001) "T cell receptor Facts Book," Academic Press, ISBN 0-12-441352-8, incorporated herein by reference in its entirety). A TCR variable region generally comprises framework regions (FR) 1, 2, 3, and 4, and complementarity determining regions (CDR) 1, 2, and 3.

[0197] As used herein, the terms "alpha chain variable region" and "Vα" are used interchangeably and refer to the variable region of the TCR alpha chain.

[0198] As used herein, the terms "β chain variable region" and "Vβ" are used interchangeably and refer to the variable region of the TCR β chain.

[0199] As used herein in the context of TCRs, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable region of a TCR chain (e.g., α or β chain). These regions are described in Lefranc, (1999) The Immunologist 7:132-136; Lefranc et al., (1999) Nucleic Acids Res 27:209-212; Lefranc (2001) "T cell receptor Facts Book." Academic Press, ISBN 0-12-441352-8; Lefranc et al., (2003) Dev Comp Immunol. 27(1):55-77; and Kabat et al., (1991) "Sequences of proteins of immunological interest," each of which is incorporated herein by reference in its entirety. In certain embodiments, the CDRs are determined according to the IMGT numbering system described in Lefranc (1999), supra. In certain embodiments, the CDRs are defined according to the Kabat numbering system as described in Kabat, supra. In certain embodiments, the CDRs are empirically defined, for example, based on structural analysis of the interaction of the TCR with a cognate antigen (e.g., a peptide or peptide-MHC complex). In certain embodiments, the α and β chain CDRs of the TCR are defined according to different conventions (e.g., according to the Kabat or IMGT numbering systems, or empirically based on structural analysis).

[0200] As used herein, the term "framework amino acid residues" refers to those amino acids within the framework region of a TCR chain (e.g., the α chain or the β chain). The term "framework region" or "FR" as used herein includes amino acid residues that are part of the TCR variable region but are not part of the CDRs.

[0201] As used herein, the term "constant region" with respect to a TCR refers to the portion of the TCR encoded by either the TRAC gene (for the TCR alpha chain) or the TRBC1 or TRBC2 genes (for the TCR beta chain), optionally lacking all or part of the transmembrane region and / or all or part of the cytoplasmic region. In certain embodiments, the TCR constant region lacks the transmembrane and cytoplasmic regions. The TCR constant region does not include amino acids encoded by the TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ genes (see, e.g., "T cell receptor Facts Book," supra).

[0202] As used herein, the terms "major histocompatibility complex" and "MHC" are used interchangeably and refer to MHC class I molecules and / or MHC class II molecules.

[0203] As used herein, the term "MHC class I" refers to a dimer of the MHC class I α chain and the β2 microglobulin chain, and the term "MHC class II" refers to a dimer of the MHC class II α chain and the MHC class II β chain.

[0204] As used herein, the terms "human leukocyte antigen" and "HLA" are used interchangeably and can also refer to proteins encoded by MHC genes. HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G refer to the major and minor gene products of MHC class I genes. HLA-DP, HLA-DQ, and HLA-DR refer to the gene products of MHC class I genes expressed on antigen presenting cells, B cells, and T cells.

[0205] As used herein, the term "peptide-MHC complex" refers to an MHC molecule (MHC class I or MHC class II) having a peptide bound to an art-recognized peptide-binding pocket of the MHC. In some embodiments, the MHC molecule is a membrane-bound protein expressed on the cell surface. In some embodiments, the MHC molecule is a soluble protein lacking a transmembrane or cytoplasmic region.

[0206] As used herein, the term "extracellular" with respect to a recombinant transmembrane protein refers to one or more portions of the recombinant transmembrane protein that are located outside the cell.

[0207] As used herein, the term "transmembrane" with respect to a recombinant transmembrane protein refers to one or more portions of the recombinant transmembrane protein that are embedded in the plasma membrane of a cell.

[0208] As used herein, the term "cytoplasmic" with respect to a recombinant transmembrane protein refers to one or more portions of the recombinant transmembrane protein that are located within the cytoplasm of a cell.

[0209] As used herein, the term "costimulatory signaling region" refers to the intracellular portion of a costimulatory molecule that is involved in mediating an intracellular signaling event.

[0210] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., TCR) and its binding partner (e.g., peptide-MHC complex). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., TCR and peptide-MHC complex). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured and / or represented in several ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and equilibrium association constant (KA). KD is calculated from the quotient of koff / kon, while KA is calculated from the quotient of koff / koff. Kon refers to the association rate constant and koff refers to the dissociation rate constant. Kon and koff can be determined by techniques known to those skilled in the art, such as using BIAcore® or KinExA. As used herein, "lower affinity" refers to a larger KD.

[0211] "Avidity" generally refers to the affinity of a binding molecule (e.g., TCR) and its binding partner (e.g., peptide-MHC complex). The binding molecules described herein can bind antigens through two (or more) sites where multiple interactions synergize to increase the "apparent" affinity. Avidity is a measure of the strength of binding between a binding molecule described herein (e.g., TCR) and an associated antigen (e.g., peptide-MHC complex). Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding molecule as well as the number of associated binding sites present on the antigen-binding molecule.

[0212] For example, "specifically binds to" may be used to refer to the ability of a TCR to preferentially bind to a particular antigen (e.g., a particular peptide or a particular peptide-MHC complex combination), as such binding is understood by one of skill in the art. For example, a TCR that specifically binds to an antigen can generally bind other antigens with lower affinity, as determined, for example, by BIAcore® or other immunoassays known in the art (see, e.g., Savage et al., (1999) Immunity. 10(4):485-92, incorporated herein by reference in its entirety). In specific embodiments, a TCR that specifically binds to an antigen binds to the antigen with an association constant (Ka) that is at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold greater than the Ka for the TCR binding to another antigen.

[0213] As used herein, "epitope" is a term in the art that refers to a localized region of an antigen (e.g., a peptide or peptide-MHC complex) to which a TCR can bind. In certain embodiments, the epitope to which a TCR binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), flow cytometry analysis, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and / or structural modeling. In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, each of which is incorporated herein by reference in its entirety). TCR:antigen crystals can be studied using well-known X-ray diffraction techniques, including X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc., see e.g., Meth Enzymol (1985) volumes 114 & 115, ed Wyckoff HW, et al., U.S. Patent Application No. 2004 / 0014194), and BUSTER (Bricogne G, (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW, and Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323, each of which is incorporated herein by reference in its entirety).Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. See, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA, (1989) Science 244:1081-1085, each of which is incorporated herein by reference in its entirety for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In a specific embodiment, the epitope of the antigen is determined using alanine scanning mutagenesis studies. In a specific embodiment, the epitope of the antigen is determined using hydrogen / deuterium exchange coupled with mass spectrometry. In a particular embodiment, the antigen is a peptide-MHC complex. In a particular embodiment, the antigen is a peptide presented by an MHC molecule.

[0214] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. In some embodiments, the method of "treatment" employs administration of a TCR or a cell expressing a TCR to a subject having, or previously diagnosed with, a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disease or disorder, or a recurrent disease or disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0215] 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.

[0216] 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.

[0217] 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, each of which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set, such as score=100, word length=12, 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, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules. (Id.) 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.

[0218] 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.

[0219] As used herein, the terms "antibody" and "antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing an antibody CDR, VH region, or VL region. Exemplary antibodies include 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), and antigen-binding fragments of 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., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0220] As used herein, the term "cistron" refers to a polynucleotide sequence from which a transgene product can be produced.

[0221] As used herein, the term "polycistronic vector" refers to a polynucleotide vector that comprises a polycistronic expression cassette.

[0222] 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).

[0223] As used herein, the term "polycistronic polynucleotide" refers to a polynucleotide that contains three or more cistrons.

[0224] 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.

[0225] As used herein, a "furin recognition site" refers to an amino acid sequence, or a nucleotide sequence encoding an amino acid sequence, that can be cleaved by the furin enzyme. The furin enzyme is also known as PACE. In some embodiments, the furin recognition site comprises the amino acid sequence RXXR (SEQ ID NO: 1), where X at position 2 is any amino acid and X at position 3 is arginine or lysine. In some embodiments, the furin recognition site comprises the sequence shown in Table 1 below.

[0226] [Table 1]

[0227] In some embodiments, the furin recognition site comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO:2 or 4, or which contains 1, 2, or 3 amino acid modifications relative to SEQ ID NO:2 or 4, or which is encoded by a polynucleotide sequence that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:3 or 5. In some embodiments, the furin recognition site, when located in a vector between a first polynucleotide sequence encoding a first protein and a second polynucleotide sequence encoding a second protein, can mediate cleavage of the first protein from the second protein (via furin), resulting in two separate polypeptides from the same mRNA molecule.

[0228] In response to recognition of the furin recognition site by the furin enzyme, the furin enzyme induces cleavage of a given polypeptide on the C-terminal side or a portion of the furin recognition site. Thus, a polypeptide produced by furin-mediated cleavage at the furin recognition site may retain all or a portion of the furin recognition site at its C-terminus. For example, the C-terminus of the first polypeptide of the present disclosure may include the amino acid sequence RAKR (SEQ ID NO: 2) or RA.

[0229] As used herein, "2A element" refers to a polynucleotide sequence that, when expressed in an mRNA, can induce ribosome skipping during translation of the mRNA in a cell. Thus, two separate polypeptides can be produced from a single mRNA molecule. The amino acid sequence encoded by the 2A element is referred to as a "self-cleaving peptide." The 2A element can be of viral origin. Exemplary 2A elements include the T2A element, the P2A element, the E2A element, and the F2A element.

[0230] As used herein, the term "P2A 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: 19 or 21, (ii) encodes the amino acid sequence of SEQ ID NO: 18 or 20, or (iii) encodes the amino acid sequence of SEQ ID NO: 18 or 20 with 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 P2A 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 the last (e.g., proline) residues at the C-terminus of the translation product of the P2A element, 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 P2A element further comprises a polynucleotide sequence at its 5' end that encodes a furin recognition site, e.g., RAKR (SEQ ID NO: 2). In some embodiments, the P2A element further comprises a polynucleotide sequence at its 5' end that encodes a furin recognition site, e.g., RAKRSGSG (SEQ ID NO: 4), and the P2A element can be referred to as a "fP2A element." In some embodiments, an fP2A element refers to a polynucleotide that (i) 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: 11, (ii) encodes the amino acid sequence of SEQ ID NO: 10, or (iii) encodes the amino acid sequence of SEQ ID NO: 10 containing one, two, or three amino acid modifications. In some embodiments, a P2A element further comprises a polynucleotide sequence at its 5' end encoding a GSG (e.g., SEQ ID NOs: 20 and 21).

[0231] 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:23 or 25, (ii) encodes the amino acid sequence of SEQ ID NO:22 or 24, or (iii) encodes the amino acid sequence of SEQ ID NO:22 or 24 with 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, for example by preventing synthesis of a peptide bond between the penultimate (e.g., glycine) and the last (e.g., proline) residues at the C-terminus of the translation product of the T2A element, 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 that encodes a furin recognition site, for example, RAKR (SEQ ID NO:2). In some embodiments, the T2A element further comprises a polynucleotide sequence at its 5' end that encodes a furin recognition site, for example, RAKRSGSG (SEQ ID NO:4), and the T2A element can be referred to as a "fT2A element." In some embodiments, a fT2A element refers to a polynucleotide that (i) 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: 13, (ii) encodes the amino acid sequence of SEQ ID NO: 12, or (iii) encodes the amino acid sequence of SEQ ID NO: 12 containing 1, 2, or 3 amino acid modifications. In some embodiments, the T2A element further comprises a polynucleotide sequence at its 5' end encoding GSG (e.g., SEQ ID NOs: 24 and 25).

[0232] 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:27 or 29, (ii) encodes the amino acid sequence of SEQ ID NO:26 or 28, or (iii) encodes the amino acid sequence of SEQ ID NO:26 or 28 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 the last (e.g., proline) residues at the C-terminus of the translation product of the F2A element, 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 F2A element further comprises a polynucleotide sequence at its 5' end encoding a furin recognition site, e.g., RAKR (SEQ ID NO:2). In some embodiments, the F2A element further comprises a polynucleotide sequence at its 5' end encoding a furin recognition site, e.g., RAKRSGSG (SEQ ID NO:4), and the F2A element can be referred to as a "fF2A element." In some embodiments, a fF2A element refers to a polynucleotide that (i) 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: 15, (ii) encodes the amino acid sequence of SEQ ID NO: 14, or (iii) encodes the amino acid sequence of SEQ ID NO: 14 containing 1, 2, or 3 amino acid modifications. In some embodiments, the F2A element further comprises a polynucleotide sequence at its 5' end encoding a GSG (e.g., SEQ ID NOs: 28 and 29).

[0233] As used herein, the term "E2A 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: 31 or 33, (ii) encodes the amino acid sequence of SEQ ID NO: 30 or 32, or (iii) encodes the amino acid sequence of SEQ ID NO: 30 or 32 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 E2A 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 the last (e.g., proline) residues at the C-terminus of the translation product of the E2A element, 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 E2A element further comprises a polynucleotide sequence at its 5' end that encodes a furin recognition site, e.g., RAKR (SEQ ID NO:2). In some embodiments, the E2A element further comprises a polynucleotide sequence at its 5' end that encodes a furin recognition site, e.g., RAKRSGSG (SEQ ID NO:4), and the E2A element can be referred to as a "fE2A element." In some embodiments, an fE2A 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: 17, (ii) encodes the amino acid sequence of SEQ ID NO: 16, or (iii) encodes the amino acid sequence of SEQ ID NO: 16 containing one, two, or three amino acid modifications. In some embodiments, an E2A element further comprises a polynucleotide sequence at its 5' end encoding GSG (e.g., SEQ ID NOs: 32 and 33).

[0234] Examples of 2A elements containing a furin recognition site at the N-terminus / 5'-terminus are shown below in Table 2. The 2A sites themselves are grouped in Table 3.

[0235] [Table 2]

[0236] [Table 3]

[0237] 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., an ITR of a 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.

[0238] 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.

[0239] 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.

[0240] 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 nucleotides in 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.

[0241] The terms "amino acid sequence" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids joined by one or more peptide bonds. As used herein, "amino acid sequence" refers to information that describes the relative order and identity of the amino acid residues that make up a polypeptide.

[0242] 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-15 protein may refer to an IL-15 protein that contains an amino acid substitution compared to a wild-type IL-15 protein that retains the ability to bind to the IL-15 receptor alpha chain (IL-15Rα). 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.

[0243] 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 IL-15 protein may refer to a fragment of the protein that retains the ability to specifically bind to IL-15Rα. Not all functions of the reference protein need be retained by a functional fragment of the protein. In some cases, one or more functions are selectively reduced or eliminated.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] As used herein, the terms "marker protein" or "marker polypeptide" are used interchangeably to refer 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 a 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-dependent cytotoxicity).

[0249] As used herein, "immune effector cells" refers to cells involved in promoting immune effector functions. 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+ T cells, natural killer T (NKT) cells), natural killer (NK) cells, B cells, mast cells, and bone marrow-derived phagocytes.

[0250] As used herein, the term "immune stem cells" refers to cells that are multipotent and can differentiate into one or more types of immune cells, including immune effector cells. Immune stem cells include, but are not limited to, bone marrow stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, umbilical cord blood stem cells, lymphoid progenitor cells, stem cell memory T cells, and stem cell memory-like T cells. In certain embodiments, immune stem cells are isolated and / or enriched from adult and fetal bone marrow, umbilical cord blood, or peripheral blood.

[0251] 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.

[0252] 5.2 T cell receptor In one aspect, the present disclosure provides a TCR that can be expressed via a polycistronic expression cassette of the present disclosure. In certain embodiments, the TCR comprises a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region, and a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ). The amino acid sequences of the constant domains contained in the TCRs disclosed herein are shown in Tables 4 and 5 below.

[0253] [Table 4-1]

[0254] [Table 4-2]

[0255] [Table 4-3]

[0256] [Table 5-1]

[0257] [Table 5-2]

[0258] As used herein, "LIV substitution" refers to a Cα sequence disclosed herein that includes a leucine residue at position 112, an isoleucine residue at position 114, and a valine residue at position 115 relative to SEQ ID NO: 40. See, e.g., SEQ ID NOs: 41 and 42. In some embodiments, independent of the LIV substitution, a Cα sequence disclosed herein can include a cysteine ​​at position 48, replacing a threonine residue. (Compare SEQ ID NOs: 40-44). In some embodiments, a Cβ sequence disclosed herein has a substitution of a cysteine ​​for a serine at residue 57. This is shown in SEQ ID NOs: 50 and 51.

[0259] Tumor protein p53 (also referred to as "p53") functions as a tumor suppressor, for example, by regulating cell division. In some embodiments, wild-type full-length p53 has the amino acid sequence of SEQ ID NO: 340, as shown below. MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO: 340)

[0260] Kirsten rat sarcoma viral oncogene homolog (KRAS), also referred to as GTPase Kras, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2, is a member of the small GTPase superfamily. There are two transcriptional variants of KRAS: KRAS variant A and KRAS variant B. Hereinafter, reference to "KRAS" (mutated or non-mutated) refers to both variant A and variant B, unless otherwise specified. In some embodiments, wild-type KRAS variant A has the amino acid sequence of SEQ ID NO: 341, and wild-type KRAS variant B has the amino acid sequence of SEQ ID NO: 342, both shown below. MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 341) MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM (SEQ ID NO: 342)

[0261] EGFR (also referred to as ERBB1 or HER1) is a transmembrane glycoprotein that belongs to the receptor tyrosine kinase (RTK) superfamily of cell surface receptors that mediate cell signaling by extracellular growth factors. Examples of wild-type (WT), non-mutated human EGFR amino acid sequences include those disclosed in GenBank Accession Nos. NP_001333826.1 (isoform e precursor), NP_001333827.1 (isoform f precursor), NP_001333828.1 (isoform g precursor), NP_001333870.1 (isoform i precursor), NP_005219.2 (isoform a precursor), NP_958439.1 (isoform b precursor), NP_958440.1 (isoform precursor), and NP_958441.1 (isoform d precursor). In some embodiments, the wild-type EGFR has the amino acid sequence of SEQ ID NO: 343.

[0262] The amino acid sequences of exemplary TCRs are shown in Table 6 herein.

[0263] [Table 6-1]

[0264] [Table 6-2]

[0265] In some embodiments, TCR001 interacts with and / or is specific for a peptide derived from the tumor protein p53 (p53). In some embodiments, the peptide is derived from a neoantigen of p53 and has the amino acid change R175H (wherein position 175 of the p53 protein is mutated from Arg to His). In some embodiments, TCR001 interacts with and / or is specific for a neoantigen in the context of HLA-A*02:01, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0266] [Table 7-1]

[0267] [Table 7-2]

[0268] In some embodiments, TCR002 interacts with and / or is specific for a peptide derived from p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR002 interacts with the neoantigen in the context of HLA-A*02:01, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0269] [Table 8-1]

[0270] [Table 8-2]

[0271] In some embodiments, TCR003 interacts with and / or is specific for a peptide derived from p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR003 interacts with the neoantigen in the context of HLA-A*02:01, as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0272] [Table 9-1]

[0273] [Table 9-2]

[0274] In some embodiments, TCR004 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR004 interacts with the neoantigen in the context of HLA-A*02:01, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0275] [Table 10-1]

[0276] [Table 10-2]

[0277] In some embodiments, TCR005 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR005 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0278] [Table 11-1]

[0279] [Table 11-2]

[0280] In some embodiments, TCR006 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR006 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0281] [Table 12-1]

[0282] [Table 12-2]

[0283] In some embodiments, TCR007 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR007 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0284] [Table 13-1]

[0285] [Table 13-2]

[0286] In some embodiments, TCR008 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR008 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0287] [Table 14-1]

[0288] [Table 14-2]

[0289] In some embodiments, TCR009 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR009 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0290] [Table 15-1]

[0291] [Table 15-2]

[0292] In some embodiments, TCR010 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR010 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0293] [Table 16-1]

[0294] [Table 16-2]

[0295] In some embodiments, TCR011 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR011 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0296] [Table 17-1]

[0297] [Table 17-2]

[0298] In some embodiments, TCR012 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR012 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0299] [Table 18-1]

[0300] [Table 18-2]

[0301] In some embodiments, TCR013 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R175H compared to the wild-type p53 sequence. In some embodiments, TCR013 interacts with a neoantigen in the context of HLA-DRB1*13:01 as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0302] [Table 19-1]

[0303] [Table 19-2]

[0304] In some embodiments, TCR014 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change Y220C compared to the wild-type p53 sequence. In some embodiments, TCR014 interacts with the neoantigen in the context of HLA-A*02:01, as described in WO2020 / 264269, which is incorporated by reference in its entirety.

[0305] [Table 20-1]

[0306] [Table 20-2]

[0307] In some embodiments, TCR015 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change Y220C compared to the wild-type p53 sequence. In some embodiments, TCR015 interacts with a neoantigen in the context of HLA-DRB1*04:01:01, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0308] [Table 21-1]

[0309] [Table 21-2]

[0310] In some embodiments, TCR016 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change Y220C compared to the wild-type p53 sequence. In some embodiments, TCR016 interacts with a neoantigen in the context of HLA-DRB3*02:02, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0311] [Table 22-1]

[0312] [Table 22-2]

[0313] In some embodiments, TCR017 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change G245S compared to the wild-type p53 sequence. In some embodiments, TCR017 interacts with a neoantigen in the context of HLA-DRB3*02:02, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0314] [Table 23-1]

[0315] [Table 23-2]

[0316] In some embodiments, TCR018 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change G245S compared to the wild-type p53 sequence. In some embodiments, TCR018 interacts with a neoantigen in the context of HLA-DRB3*02:02, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0317] [Table 24-1]

[0318] [Table 24-2]

[0319] In some embodiments, TCR019 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change G245S compared to the wild-type p53 sequence. In some embodiments, TCR019 interacts with a neoantigen in the context of HLA-DRB3*02:02, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0320] [Table 25-1]

[0321] [Table 25-2]

[0322] In some embodiments, TCR020 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change G245S compared to the wild-type p53 sequence. In some embodiments, TCR020 interacts with the neoantigen in the context of HLA-DRB3*02:02, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0323] [Table 26-1]

[0324] [Table 26-2]

[0325] In some embodiments, TCR021 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR021 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0326] [Table 27-1]

[0327] [Table 27-2]

[0328] In some embodiments, TCR022 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D compared to the wild-type KRAS sequence. In some embodiments, TCR022 interacts with a neoantigen in the context of HLA-A*11:01 as described in WO2021 / 163434, which is incorporated by reference in its entirety.

[0329] [Table 28-1]

[0330] [Table 28-2]

[0331] In some embodiments, TCR023 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR023 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0332] [Table 29-1]

[0333] [Table 29-2]

[0334] In some embodiments, TCR024 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR024 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0335] [Table 30-1]

[0336] [Table 30-2]

[0337] In some embodiments, TCR025 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR025 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0338] [Table 31-1]

[0339] [Table 31-2]

[0340] In some embodiments, TCR026 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR026 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0341] [Table 32-1]

[0342] [Table 32-2]

[0343] In some embodiments, TCR027 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR027 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0344] [Table 33-1]

[0345] [Table 33-2]

[0346] In some embodiments, TCR028 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR028 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0347] [Table 34-1]

[0348] [Table 34-2]

[0349] In some embodiments, TCR029 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR029 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0350] [Table 35-1]

[0351] [Table 35-2]

[0352] In some embodiments, TCR030 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR030 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0353] [Table 36-1]

[0354] [Table 36-2]

[0355] In some embodiments, TCR031 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR031 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0356] [Table 37-1]

[0357] [Table 37-2]

[0358] In some embodiments, TCR032 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR032 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0359] [Table 38-1]

[0360] [Table 38-2]

[0361] In some embodiments, TCR034 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR034 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0362] [Table 39-1]

[0363] [Table 39-2]

[0364] In some embodiments, TCR034 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR034 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0365] [Table 40-1]

[0366] [Table 40-2]

[0367] In some embodiments, TCR035 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR035 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0368] [Table 41-1]

[0369] [Table 41-2]

[0370] In some embodiments, TCR036 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR036 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0371] [Table 42-1]

[0372] [Table 42-2]

[0373] In some embodiments, TCR037 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR037 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0374] [Table 43-1]

[0375] [Table 43-2]

[0376] In some embodiments, TCR038 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR038 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0377] [Table 44-1]

[0378] [Table 44-2]

[0379] In some embodiments, TCR039 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR039 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0380] [Table 45-1]

[0381] [Table 45-2]

[0382] In some embodiments, TCR040 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR040 interacts with the neoantigen in the context of HLA-A*02:01, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0383] [Table 46-1]

[0384] [Table 46-2]

[0385] In some embodiments, TCR041 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR041 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0386] [Table 47-1]

[0387] [Table 47-2]

[0388] In some embodiments, TCR042 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR042 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0389] [Table 48-1]

[0390] [Table 48-2]

[0391] In some embodiments, TCR043 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR043 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0392] [Table 49-1]

[0393] [Table 49-2]

[0394] In some embodiments, TCR044 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR044 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0395] [Table 50-1]

[0396] [Table 50-2]

[0397] In some embodiments, TCR045 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR045 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0398] [Table 51-1]

[0399] [Table 51-2]

[0400] In some embodiments, TCR046 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR046 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0401] [Table 52-1]

[0402] [Table 52-2]

[0403] In some embodiments, TCR047 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR047 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0404] [Table 53-1]

[0405] [Table 53-2]

[0406] In some embodiments, TCR048 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248Q compared to the wild-type p53 sequence. In some embodiments, TCR048 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0407] [Table 54-1]

[0408] [Table 54-2]

[0409] In some embodiments, TCR049 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild-type p53 sequence. In some embodiments, TCR049 interacts with a neoantigen in the context of HLA-A*68:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0410] [Table 55-1]

[0411] [Table 55-2]

[0412] In some embodiments, TCR050 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild-type p53 sequence. In some embodiments, TCR050 interacts with the neoantigen in the context of HLA-A*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0413] [Table 56-1]

[0414] [Table 56-2]

[0415] In some embodiments, TCR051 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild type p53 sequence. In some embodiments, TCR051 interacts with a neoantigen in the context of HLA-DPA1*03:01 / DPB1*02:01:02, as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0416] [Table 57-1]

[0417] [Table 57-2]

[0418] In some embodiments, TCR052 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild-type p53 sequence. In some embodiments, TCR052 interacts with the neoantigen in the context of HLA-A*68:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0419] [Table 58-1]

[0420] [Table 58-2]

[0421] In some embodiments, TCR053 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild-type p53 sequence. In some embodiments, TCR053 interacts with the neoantigen in the context of HLA-A*68:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0422] [Table 59-1]

[0423] [Table 59-2]

[0424] In some embodiments, TCR054 interacts with and / or is specific for p53. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild type p53 sequence. In some embodiments, TCR054 interacts with a neoantigen in the context of DPA1*01:03 / DBP1*02:01 as described in WO2019 / 067243, which is incorporated by reference in its entirety.

[0425] [Table 60-1]

[0426] [Table 60-2]

[0427] In some embodiments, TCR055 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR055 interacts with the neoantigen in the context of HLA-C*01:02 as described in WO2021 / 163477, which is incorporated by reference in its entirety.

[0428] [Table 61-1]

[0429] [Table 61-2]

[0430] In some embodiments, TCR056 interacts with p53 and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild-type p53 sequence. In some embodiments, TCR056 interacts with a neoantigen in the context of HLA-A*02:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0431] [Table 62-1]

[0432] [Table 62-2]

[0433] In some embodiments, TCR057 interacts with p53 and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of p53. In some embodiments, the neoantigen has the amino acid change R248W compared to the wild-type p53 sequence. In some embodiments, TCR057 interacts with a neoantigen in the context of HLA-A*68:01 as described in WO 2019 / 067243, which is incorporated by reference in its entirety.

[0434] [Table 63-1]

[0435] [Table 63-2]

[0436] In some embodiments, TCR058 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR058 interacts with a neoantigen in the context of HLA-C*01:02, as described in WO2021 / 163477, which is incorporated by reference in its entirety.

[0437] [Table 64-1]

[0438] [Table 64-2]

[0439] In some embodiments, TCR059 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR059 interacts with the neoantigen in the context of HLA-C*01:02 as described in WO2021 / 163477, which is incorporated by reference in its entirety.

[0440] [Table 65-1]

[0441] [Table 65-2]

[0442] In some embodiments, TCR060 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR060 interacts with a neoantigen in the context of the HLA-DPA1*01:03 and HLA-DPB1*03:01 chains as described in WO2021 / 173902, which is incorporated by reference in its entirety.

[0443] [Table 66-1]

[0444] [Table 66-2]

[0445] In some embodiments, TCR061 interacts with and / or is specific for the tumor protein KRAS (KRAS). In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12C compared to the wild type KRAS sequence. In some embodiments, TCR061 interacts with the neoantigen in the context of HLA-DRB1*11:01 as described in WO2019 / 060349, which is incorporated by reference in its entirety.

[0446] [Table 67-1]

[0447] [Table 67-2]

[0448] In some embodiments, TCR062 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D compared to the wild type KRAS sequence. In some embodiments, TCR062 interacts with a neoantigen in the context of HLA-C*08:02, as described in WO 2018 / 026691, which is incorporated by reference in its entirety.

[0449] [Table 68-1]

[0450] [Table 68-2]

[0451] In some embodiments, TCR063 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D compared to the wild type KRAS sequence. In some embodiments, TCR063 interacts with a neoantigen in the context of HLA-C*08:02 as described in WO 2018 / 026691, which is incorporated by reference in its entirety.

[0452] [Table 69-1]

[0453] [Table 69-2]

[0454] In some embodiments, TCR064 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D compared to the wild type KRAS sequence. In some embodiments, TCR064 interacts with a neoantigen in the context of HLA-C*08:02, as described in WO 2018 / 026691, which is incorporated by reference in its entirety.

[0455] [Table 70-1]

[0456] [Table 70-2]

[0457] In some embodiments, TCR065 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D compared to the wild type KRAS sequence. In some embodiments, TCR065 interacts with a neoantigen in the context of HLA-Cw*08:02 as described in WO 2017 / 048593, which is incorporated by reference in its entirety.

[0458] [Table 71-1]

[0459] [Table 71-2]

[0460] In some embodiments, TCR066 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D compared to the wild type KRAS sequence. In some embodiments, TCR066 interacts with a neoantigen in the context of HLA-C*08:02 as described in WO 2018 / 026691, which is incorporated by reference in its entirety.

[0461] [Table 72-1]

[0462] [Table 72-2]

[0463] In some embodiments, TCR067 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D and / or the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR067 interacts with a neoantigen in the context of HLA-A11 as described in WO2016 / 085904, which is incorporated by reference in its entirety.

[0464] [Table 73-1]

[0465] [Table 73-2]

[0466] In some embodiments, TCR068 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D and / or the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR068 interacts with a neoantigen in the context of HLA-A11 as described in WO2016 / 085904, which is incorporated by reference in its entirety.

[0467] [Table 74-1]

[0468] [Table 74-2]

[0469] In some embodiments, TCR069 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D and / or the amino acid change G12V compared to the wild type KRAS sequence. In some embodiments, TCR069 interacts with a neoantigen in the context of HLA-A11 as described in WO2016 / 085904, which is incorporated by reference in its entirety.

[0470] [Table 75-1]

[0471] [Table 75-2]

[0472] In some embodiments, TCR070 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D and / or the amino acid change G12V compared to the wild type KRAS sequence. In some embodiments, TCR070 interacts with the neoantigen in the context of HLA-A11 as described in WO2016 / 085904, which is incorporated by reference in its entirety.

[0473] [Table 76-1]

[0474] [Table 76-2]

[0475] [Table 76-3]

[0476] In some embodiments, TCR071 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12D and / or the amino acid change G12V compared to the wild type KRAS sequence. In some embodiments, TCR071 interacts with a neoantigen in the context of HLA-A11 as described in WO2016 / 085904, which is incorporated by reference in its entirety.

[0477] [Table 77-1]

[0478] [Table 77-2]

[0479] In some embodiments, TCR072 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12R compared to the wild type KRAS sequence. In some embodiments, TCR072 interacts with the neoantigen in the context of the HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer as described in WO2020 / 154275, which is incorporated by reference in its entirety.

[0480] [Table 78-1]

[0481] [Table 78-2]

[0482] In some embodiments, TCR073 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12R compared to the wild-type KRAS sequence. In some embodiments, TCR073 interacts with the neoantigen in the context of the HLA-DRB5*01:HLA-DRA*01:01 heterodimer, as described in WO2020 / 154275, which is incorporated by reference in its entirety.

[0483] [Table 79-1]

[0484] [Table 79-2]

[0485] In some embodiments, TCR074 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR074 interacts with the neoantigen in the context of an HLA-A3 heterodimer, as described in WO2020 / 086827, which is incorporated by reference in its entirety.

[0486] [Table 80-1]

[0487] [Table 80-2]

[0488] In some embodiments, TCR075 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild type KRAS sequence. In some embodiments, TCR075 interacts with a neoantigen in the context of HLA-A*11:01 as described in WO 2019 / 112941, which is incorporated by reference in its entirety.

[0489] [Table 81-1]

[0490] [Table 81-2]

[0491] In some embodiments, TCR076 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild type KRAS sequence. In some embodiments, TCR076 interacts with a neoantigen in the context of HLA-DRB1*07:01 as described in WO2019 / 060349, which is incorporated by reference in its entirety.

[0492] [Table 82-1]

[0493] [Table 82-2]

[0494] In some embodiments, TCR077 interacts with epidermal growth factor receptor (EGFR) oncoprotein and / or is specific for KRAS. In some embodiments, the peptide is derived from an EGFR neoantigen. In some embodiments, the neoantigen has the amino acid changes E746-A750del compared to the wild-type EGFR sequence. In some embodiments, TCR077 interacts with the neoantigen in the context of a heterodimer of HLA-DPA1*02:01 and HLA-DPB1*01:01, as described in WO 2019 / 213195, which is incorporated by reference in its entirety.

[0495] [Table 83-1]

[0496] [Table 83-2]

[0497] In some embodiments, TCR078 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR078 interacts with a neoantigen in the context of the HLA-DPA1*01:03 and HLA-DPB1*03:01 chains as described in WO2021 / 173902, which is incorporated by reference in its entirety.

[0498] [Table 84-1]

[0499] [Table 84-2]

[0500] In some embodiments, TCR079 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild type KRAS sequence. In some embodiments, TCR079 interacts with a neoantigen in the context of the HLA-DPA1*01:03 and HLA-DPB1*03:01 chains as described in WO2021 / 173902, which is incorporated by reference in its entirety.

[0501] [Table 85-1]

[0502] [Table 85-2]

[0503] In some embodiments, TCR080 interacts with and / or is specific for KRAS. In some embodiments, the peptide is derived from a neoantigen of KRAS. In some embodiments, the neoantigen has the amino acid change G12V compared to the wild-type KRAS sequence. In some embodiments, TCR080 interacts with a neoantigen in the context of the HLA-DPA1*01:03 and HLA-DPB1*03:01 chains as described in WO2021 / 173902, which is incorporated by reference in its entirety.

[0504] The present disclosure also provides for the use of other TCR V alpha and beta sequences, and any other alpha or beta chains, in the polycistronic vectors, engineered cells, or pharmaceutical compositions described herein. These TCR V alpha and beta sequences, and alpha or beta chains, are described in International Publication Nos. WO2016 / 085904, WO2017 / 048593, WO2018 / 026691, WO2019 / 060349, WO2019 / 067243, WO2019 / 070435, WO2019 / 11294, and the like. 1, WO2019 / 213195, WO2020 / 086827, WO2020 / 154275, WO2020 / 264269, WO2021 / 163434, WO2021 / 163477, and WO2021 / 173902, all of which are incorporated by reference in their entireties.

[0505] The CDRs of the TCRs disclosed herein may be defined using any skilled artisan recognized numbering convention. Additionally or alternatively, the CDRs may be empirically defined, for example, based on structural analysis of the interaction of the TCR with a cognate antigen (e.g., a peptide or peptide-MHC complex). In some embodiments, the CDR3 of the TCR may further comprise an N-terminal cysteine ​​and / or a C-terminal phenylalanine or tryptophan.

[0506] The TCRs disclosed herein can be used in any TCR structural format. For example, in certain embodiments, the TCR is a full-length TCR, including a full-length α chain and a full-length β chain. The transmembrane region (and optionally the cytoplasmic region) can be removed from the full-length TCR to generate a soluble TCR. Thus, in certain embodiments, the TCR is a soluble TCR that lacks the transmembrane region and / or the cytoplasmic region. Methods for generating soluble TCRs are well known in the art. In some embodiments, the soluble TCR contains engineered disulfide bonds that promote dimerization, see, e.g., U.S. Patent No. 7,329,731 (incorporated herein by reference in its entirety). In some embodiments, soluble TCRs are generated by fusing the extracellular domain of a TCR described herein to other protein domains, such as maltose binding protein, thioredoxin, human constant kappa domain, or leucine zipper (see, e.g., Loset et al., Front Oncol. 2014;4:378, incorporated herein by reference in its entirety). Single chain TCRs (scTCRs) can also be generated that include Vα and Vβ linked by a peptide linker. Such scTCRs can include Vα and Vβ, each linked to a TCR constant region. Alternatively, scTCRs can include Vα and Vβ, where Vα, Vβ, or both Vα and Vβ are not linked to a TCR constant region. Exemplary scTCRs are described in PCT Publication Nos. 2003 / 020763, 2004 / 033685, and 2011 / 044186, each of which is incorporated herein by reference in its entirety. Additionally, the TCRs disclosed herein may comprise two polypeptide chains (e.g., an α chain and a β chain) that are engineered such that each chain has a cysteine ​​residue capable of forming an interchain disulfide bond. Thus, in certain embodiments, the TCRs disclosed herein comprise two polypeptide chains linked by an engineered disulfide bond.Exemplary TCRs with engineered disulfide bonds are described in US Pat. Nos. 8,361,794 and 8,906,383, each of which is incorporated herein by reference in its entirety.

[0507] In certain embodiments, the TCRs disclosed herein comprise one or more chains (e.g., an α chain and / or a β chain) with a transmembrane region. In certain embodiments, the TCRs disclosed herein comprise two chains (e.g., an α chain and a β chain) with a transmembrane region. The transmembrane region may be the endogenous transmembrane region of that TCR chain, a variant of the endogenous transmembrane region, or a heterologous transmembrane region. In certain embodiments, the TCRs disclosed herein comprise an α chain and a β chain with an endogenous transmembrane region.

[0508] In certain embodiments, the TCRs disclosed herein comprise one or more chains (e.g., an α chain and / or a β chain) that have a cytoplasmic region. In certain embodiments, the TCRs disclosed herein comprise two chains (e.g., an α chain and a β chain), each having a cytoplasmic region. The cytoplasmic region may be the endogenous cytoplasmic region of that TCR chain, a variant of the endogenous cytoplasmic region, or a heterologous cytoplasmic region. In certain embodiments, the TCRs disclosed herein comprise two chains (e.g., an α chain and a β chain), both of which have a transmembrane region, but one chain lacks a cytoplasmic region. In certain embodiments, the TCRs disclosed herein comprise two chains (e.g., an α chain and a β chain), both of which have an endogenous transmembrane region, but lack an endogenous cytoplasmic region. In certain embodiments, the TCRs disclosed herein comprise an α chain and a β chain, both of which have an endogenous transmembrane region, but lack an endogenous cytoplasmic region. In certain embodiments, the TCRs disclosed herein comprise a costimulatory signaling region from a costimulatory molecule. See, e.g., PCT Publication Nos. 1996 / 018105, 1999 / 057268, and 2000 / 031239, and U.S. Patent No. 7,052,906, all of which are incorporated by reference in their entireties.

[0509] In certain embodiments, the present disclosure provides a polypeptide comprising a TCR α chain variable region (Vα) and a β chain variable region (Vβ) fused together. For example, such a polypeptide may comprise Vα and Vβ, or Vβ and Vα, in order, optionally with a linker (e.g., a peptide linker) between the two regions. For example, a furin and / or 2A cleavage site (e.g., one of the sequences in Tables 2 or 3), or a combination thereof, may be used in the linker of a Vα / Vβ fusion polypeptide. In certain embodiments, the present disclosure provides a polypeptide comprising a TCR α chain and a β chain fused together. For example, such a polypeptide may comprise an α chain and a β chain, or a β chain and an α chain, in order, optionally with a linker (e.g., a peptide linker) between the two chains. For example, a furin and / or 2A cleavage site (e.g., one of the sequences in Tables 2 or 3), or a combination thereof, may be used in the linker of an α / β fusion polypeptide. For example, a fusion polypeptide may comprise, from N-terminus to C-terminus, an alpha chain of the TCR, a furin cleavage site, a 2A cleavage site, and a beta chain of the TCR. In certain embodiments, the polypeptide comprises, from N-terminus to C-terminus, a beta chain of the TCR, a furin cleavage site, a 2A element, and an alpha chain of the TCR.

[0510] 5.3 How to use In another aspect, the present disclosure provides a method of treating a subject using a polycistronic polynucleotide, a recombinant vector, an engineered cell (e.g., a cell comprising a heterologous and / or recombinant nucleic acid), or a pharmaceutical composition disclosed herein. Any disease or disorder in a subject that would benefit from treatment with a recombinant cell of the present disclosure, or a polynucleotide or vector of the present disclosure, can be treated using the methods disclosed herein.

[0511] In certain embodiments, the methods comprise administering to a subject an effective amount of a recombinant cell or population thereof as disclosed herein.

[0512] As disclosed below, the cells administered to the subject can be autologous or allogeneic to the subject. In certain embodiments, autologous cells are obtained from a cancer patient immediately after cancer treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo immediately after treatment during the period when the patient is usually recovering from the treatment. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, in certain embodiments, cells are collected from blood, bone marrow, lymph nodes, thymus, or another tissue or fluid, or apheresis product during this recovery period. Furthermore, in certain aspects, mobilization and conditioning regimens can be used to create a favorable state in the subject for repopulation, recirculation, regeneration, and / or expansion of certain cell types, particularly within a defined time frame after therapy.

[0513] The number of cells used will depend on many circumstances, including the lifespan of the cells, the protocol used (e.g., number of administrations), the proliferation potential of the cells, the stability of the recombinant construct, etc. In certain embodiments, the cells are applied as a dispersion and generally injected at or near the site of interest. The cells may be administered in any physiologically acceptable medium.

[0514] In certain embodiments, the cancer is lung cancer, bile duct cancer (e.g., cholangiocarcinoma), pancreatic cancer, colorectal cancer, ovarian cancer, or gynecological cancer. In certain embodiments, the cancer is leukemia (e.g., mixed lineage leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, or chronic myeloid leukemia), alveolar rhabdomyosarcoma, bone cancer, brain tumor (e.g., glioma, e.g., glioblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma), joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, myeloma (e.g., chronic myeloid cancer), colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, or rectal cancer. Cancer, gastrointestinal cancer-like tumors, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, retinal, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), gastric cancer, small intestine cancer, soft tissue cancer, gastric cancer, cancer, sarcoma (e.g., colon sarcoma, rhabdomyosarcoma), skin cancer, testicular cancer, thyroid cancer, head and neck cancer, ureter cancer, urinary bladder cancer. In certain embodiments, the cancer is melanoma, breast cancer, lung cancer, prostate cancer, thyroid cancer, ovarian cancer, or synovial sarcoma. In one embodiment, the cancer is synovial sarcoma or liposarcoma (e.g., myxoid / round cell liposarcoma). In certain embodiments, the cancer is lung cancer, bile duct cancer, pancreatic cancer, colorectal cancer, gynecological cancer, or ovarian cancer.

[0515] The polycistronic polynucleotides, recombinant vectors, engineered cells, or pharmaceutical compositions described herein may be delivered to a subject by a variety of routes, including, but not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intrathecal, and subcutaneous routes. Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In certain embodiments, the polycistronic polynucleotides, recombinant vectors, engineered cells, or pharmaceutical compositions described herein are delivered intravenously. In certain embodiments, the polycistronic polynucleotides, vectors, engineered cells, or pharmaceutical compositions described herein are delivered subcutaneously. In certain embodiments, the polycistronic polynucleotides, recombinant vectors, engineered cells, or pharmaceutical compositions described herein are delivered intratumorally. In certain embodiments, the polycistronic polynucleotides, recombinant vectors, engineered cells, or pharmaceutical compositions described herein are delivered to a tumor-draining lymph node.

[0516] The amount of the polycistronic polynucleotide, recombinant vector, engineered cell, or pharmaceutical composition that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0517] The exact dose to be used in the composition also depends on the route of administration and the severity of the infection or disease caused thereby, and should be determined according to the judgment of the practitioner and the circumstances of each subject.For example, the effective dose may also vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight, and health), whether the patient is a human or an animal, other medicines administered, or whether the treatment is preventive or therapeutic.Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated.Therapeutic dosages can be optimally titrated to optimize safety and efficacy.

[0518] 5.4 IL-15 / IL-15Rα fusion protein The present disclosure also provides a recombinant vector comprising a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is membrane-bound. In some embodiments, the cytokine is a fusion protein comprising a soluble cytokine, or a functional fragment or variant thereof, operably linked to the cytokine's cognate receptor, or a functional fragment or variant thereof, optionally in its membrane-bound form. In some embodiments, the fusion protein comprises human IL-15 (hIL-15) operably linked to human IL-15Rα (hIL-15Rα). In the membrane-bound form, this fusion protein is 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.

[0519] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 81, 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: 81. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the amino acids of the linker consist of the amino acid sequence of SEQ ID NO: 81, 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: 81. In some embodiments, the amino acids of the linker consist of the amino acid sequence of SEQ ID NO: 81.

[0520] In some embodiments, the linker is encoded by a polynucleotide sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO:82. In some embodiments, the linker is encoded by a polynucleotide sequence of SEQ ID NO:82. In some embodiments, the 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:76. In some embodiments, the hIL-15 comprises the amino acid sequence of SEQ ID NO:76. 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:76. In some embodiments, the amino acid sequence of hIL-15 consists of the amino acid sequence of SEQ ID NO:76.

[0521] In some embodiments, hIL-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: 77. In some embodiments, hIL-15 is encoded by the polynucleotide sequence of SEQ ID NO:77.

[0522] In some embodiments, hIL-15Rα 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: 78. In some embodiments, hIL-15Rα comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the amino acid sequence of hIL-15Rα 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 hIL-15Rα consists of the amino acid sequence of SEQ ID NO: 78.

[0523] 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: 79. In some embodiments, hIL-15Rα is encoded by the polynucleotide sequence of SEQ ID NO:79.

[0524] 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:70 or 73. 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:70. 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:73. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:70 or 73. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:70. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:73.

[0525] 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:70 or 73. 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:70. 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:73. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO:70 or 73. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO:70. In some embodiments, the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO:73.

[0526] 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: 71 or 74. 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: 71. 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: 74.

[0527] In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 71 or 74. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 71. In some embodiments, the fusion protein is encoded by the polynucleotide sequence of SEQ ID NO: 74.

[0528] Exemplary cytokine fusion proteins and components thereof are disclosed in Table 7. 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.

[0529] The amino acid and polynucleotide sequences of exemplary cytokine fusion proteins and component polypeptides are provided in Table 7 herein.

[0530] [Table 86-1]

[0531] [Table 86-2]

[0532] [Table 86-3]

[0533] [Table 86-4]

[0534] 5.5 Marker Proteins The marker proteins described herein function to allow for the selective depletion of cells contacted with a recombinant vector (e.g., recombinant cell) disclosed herein 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 the recombinant cell. In some embodiments, the marker protein is expressed on the surface of the recombinant cell.

[0535] 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 recombinant cells by administering an antibody that recognizes the hHER1 marker protein expressed on the surface of the recombinant cells. An exemplary antibody that binds to the hHER1 marker protein is cetuximab.

[0536] 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.

[0537] In some embodiments, domain III of hHER1 comprises the amino acid sequence of SEQ ID NO: 104, or the amino acid sequence of SEQ ID NO: 104 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: 104, or the amino acid sequence of SEQ ID NO: 10 with one, two, or three amino acid modifications.

[0538] 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: 105. 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.

[0539] 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.

[0540] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 108, 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: 108. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 108, 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: 108. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 108.

[0541] 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:100, 103, 112, or 113. 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:100. 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:112. 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:113.

[0542] In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 100 or 103. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the marker protein comprises the amino acid sequence of SEQ ID NO: 103.

[0543] 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:100, 103, 112, or 113. 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:100. 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:112. 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:113.

[0544] In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 100, 103, 112, or 113. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 100. 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: 112. In some embodiments, the marker protein consists of the amino acid sequence of SEQ ID NO: 113.

[0545] 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 recombinant cells by administering an antibody that recognizes the hCD20 marker protein expressed on the surface of the recombinant cells. An exemplary antibody that binds to the hCD20 marker protein is rituximab.

[0546] Amino acid sequences of exemplary marker proteins are provided in Table 8 herein.

[0547] [Table 87-1]

[0548] [Table 87-2]

[0549] 5.6 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. In some embodiments, the polycistronic expression cassette comprises six cistrons.

[0550] In some embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministrings, and oligonucleotides (e.g., mRNA, naked DNA). In some embodiments, the polycistronic vector is a DNA plasmid vector.

[0551] 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 viral vectors (e.g., HSV1, HSV2), alphaviral vectors (e.g., SFV, SIN, VEE, M1), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdoviral vectors (e.g., rabies virus, VSV), measles viral vectors (e.g., MV-Edm), Newcastle disease viral vectors, pox viral vectors (e.g., VV), measles virus, and picornaviral vectors (e.g., coxsackie virus).

[0552] 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.

[0553] 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.

[0554] 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: 150. In some embodiments, the promoter comprises a polynucleotide sequence of SEQ ID NO: 150. 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: 150. In some embodiments, the promoter polynucleotide sequence consists of the polynucleotide sequence of SEQ ID NO: 150.

[0555] 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: 151. In some embodiments, the polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 151. 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: 151. In some embodiments, the polyA sequence consists of the nucleic acid sequence of SEQ ID NO: 151.

[0556] Polynucleotide sequences of exemplary promoters and polyA sequences are provided in Table 9 herein.

[0557] [Table 88]

[0558] In some embodiments, the polycistronic expression cassette comprises a polynucleotide sequence encoding an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence listed in Tables 10A-10C.

[0559] [Table 89-1]

[0560] [Table 89-2]

[0561] [Table 89-3]

[0562]

Table 89-4

[0563]

Table 89-5

[0564]

Table 89-6

[0565]

Table 90-1

[0566]

Table 90-2

[0567]

Table 90-3

[0568]

Table 90-4

[0569]

Table 91-1

[0570]

Table 91-2

[0571] Tables 11A, B, and C below provide exemplary polynucleotide sequences for use in constructing vectors of the present disclosure. As shown in Tables 11A, B, and C, vectors of the present disclosure can include one or more of the following sequences: (1) an "AP" sequence that encodes (i) a Cα sequence disclosed herein and (ii) a P2A element sequence disclosed herein, (2) a "BT" sequence that encodes (i) a Cβ sequence disclosed herein and (ii) a T2A element sequence disclosed herein, (3) a "BT15" ​​sequence that encodes (i) a Cβ sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) an mbIL15 sequence disclosed herein, (4) a "BT15" ​​sequence that encodes (i) a Cα sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) a mbIL15 sequence disclosed herein, (5) a "BT15" ​​sequence that encodes (i) a Cα sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) a mbIL15 sequence disclosed herein, (6) a "BT15" ​​sequence that encodes (i) a Cβ sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) a mbIL15 sequence disclosed herein, (7) a "BT15" ​​sequence that encodes (i) a Cα sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) a mbIL15 sequence disclosed herein, (8) a "BT15" ​​sequence that encodes (i) a Cβ sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) a mbIL15 sequence disclosed herein, (9) a "BT15" ​​sequence that encodes (i) a Cβ sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) a mbIL15 sequence disclosed herein, (5) a "BP" sequence encoding (i) a Cβ sequence disclosed herein, and (ii) a P2A element sequence disclosed herein; (6) a "BP15" sequence encoding (i) a Cβ sequence disclosed herein, (ii) a P2A element sequence disclosed herein, and (iii) an mbIL15 sequence disclosed herein; (7) (i) a Cα sequence disclosed herein, (ii) a P2A element sequence disclosed herein, and (iii) a "BP15" sequence encoding (i) a Cα sequence disclosed herein, (ii) a P2A element sequence disclosed herein; (8) (i) the mbIL15 sequence disclosed herein, (ii) a "15T" sequence encoding a T2A element sequence disclosed herein, (9) (i) a Cα sequence disclosed herein, (ii) a P2A element sequence disclosed herein, (iii) the mbIL15 sequence disclosed herein, and (iv) an "AP15T" sequence encoding a T2A element sequence disclosed herein, (10) (i) an "AT15" sequence encoding a Cα sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) an mbIL15 sequence disclosed herein; (11) (i) an mbIL15 sequence disclosed herein, and (ii) a "15P" sequence encoding a P2A element sequence disclosed herein; (12) (i) a Cα sequence disclosed herein, (ii) a T2A element sequence disclosed herein, and (iii) an mbIL15 sequence disclosed herein;and (iv) an "AT15P" sequence encoding a P2A element sequence disclosed herein; (13) a "BP15T" sequence encoding (i) a Cβ sequence disclosed herein; (ii) a P2A element sequence disclosed herein; (iii) an mbIL15 sequence disclosed herein; and (iv) a T2A element sequence disclosed herein; (14) a "BT15P" sequence encoding (i) a Cβ sequence disclosed herein; (ii) a T2A element sequence disclosed herein; (iii) an mbIL15 sequence disclosed herein; and (iv) a P2A element sequence disclosed herein.

[0572] The nucleotide sequences (and their corresponding amino acid sequences) provided herein may be used in any suitable combination. A "suitable combination" is one in which a desired molecular function is provided by one or more of the sequences disclosed herein. For example, in general, any 2A element sequence provided herein can provide the functions of ribosome skipping (via the 2A element) and, optionally, furin-mediated cleavage (via the furin recognition site). Thus, the "AT" sequence in the vector of the present disclosure may be replaced by the "AP" sequence of the present disclosure in alternative embodiments. Similarly, "AE" and "AF" sequences, which include Cα region sequences and E2A or F2A element sequences, may also be used. "BT", "BP", "BE", and "BF" sequences, which include Cβ region sequences and 2A element sequences, are also all interchangeable. "15T", "15P", "15E", and "15F" sequences, which include mbIL15 sequences and 2A element sequences, are also all interchangeable. Additionally, any combination of TCR alpha, TCR beta, and mbIL15 sequences may appear in any order 5' to 3' on the vectors of the present disclosure and may be separated by sequences that provide appropriate 2A element sequence function (e.g., ribosomal skipping, furin cleavage).

[0573] Thus, the sequences of the disclosure provide ribosomal skipping, furin recognition, TCR alpha function, TCR beta function, and mbIL15 function in any suitable combination or order from 5' to 3'.

[0574]

Table 92-1

[0575]

Table 92-2

[0576]

Table 92-3

[0577]

Table 92-4

[0578]

Table 92-5

[0579]

Table 92-6

[0580]

Table 92-7

[0581]

Table 92-8

[0582]

Table 92-9

[0583]

Table 92-10

[0584]

Table 92-11

[0585]

Table 92-12

[0586]

Table 93-1

[0587]

Table 93-2

[0588]

Table 93-3

[0589]

Table 93-4

[0590]

Table 93-5

[0591]

Table 93-6

[0592]

Table 93-7

[0593]

Table 93-8

[0594] [Table 93-9]

[0595] [Table 94-1]

[0596] [Table 94-2]

[0597] [Table 94-3]

[0598] [Table 94-4]

[0599] [Table 94-5]

[0600] 5.7 Transposon and Transposase Systems In some embodiments, the transgene of the recombinant vector is introduced into the immune effector cells via a synthetic DNA transposable element, e.g., a DNA transposon / transposase system, e.g., 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.

[0601] 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 piggyBac 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 piggyBat 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 piggyBat 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).Additional 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.

[0602] 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.

[0603] 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.

[0604] 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.

[0605] 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: 290 or 291. 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: 290. In some embodiments, the left ITR comprises a polynucleotide sequence of SEQ ID NO: 290. 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: 291. In some embodiments, the left ITR comprises the polynucleotide sequence of SEQ ID NO: 291. 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: 292, 293, or 294. 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: 292. 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: 293. 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: 294. In some embodiments, the right ITR comprises the polynucleotide sequence of SEQ ID NO: 292. In some embodiments, the right ITR comprises the polynucleotide sequence of SEQ ID NO: 293. In some embodiments, the right ITR comprises the polynucleotide sequence of SEQ ID NO: 294.

[0606] Exemplary polynucleotide sequences of SB ITRs are provided in Table 12 herein.

[0607] [Table 95-1]

[0608] [Table 95-2]

[0609] 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.

[0610] 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: 300. In some embodiments, the DNA transposase comprises the amino acid sequence of SEQ ID NO: 300. 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: 300. In some embodiments, the amino acid sequence of the DNA transposase consists of the amino acid sequence of SEQ ID NO: 300.

[0611] 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: 300, lacking its N-terminal methionine, i.e., amino acids 2-340 of SEQ ID NO: 300. In some embodiments, the DNA transposase comprises an amino acid sequence of SEQ ID NO: 300, lacking its N-terminal methionine, i.e., amino acids 2-340 of SEQ ID NO: 300. 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: 300, lacking its N-terminal methionine, i.e., amino acids 2-340 of SEQ ID NO: 300. In some embodiments, the amino acid sequence of the DNA transposase consists of the amino acid sequence of SEQ ID NO:300, lacking its N-terminal methionine, i.e., amino acids 2 to 340 of SEQ ID NO:300.

[0612] 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: 301. In some embodiments, the DNA transposase is encoded by the polynucleotide sequence of SEQ ID NO:301.

[0613] 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.

[0614] Exemplary SB transposase amino acid and polynucleotide sequences are provided in Table 13 herein.

[0615] [Table 96]

[0616] 5.8 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. For example, in certain embodiments, the T cell is selected from the group consisting of naive T cells (CD4+ or CD8+), killer CD8+ T cells, cytotoxic CD4+ T cells, CD4+ T cells corresponding to Th1, Th2, Th9, Th17, Th22, follicular helper (Tfh), regulatory (Treg) lineages, CD8+ cytotoxic T cells, CD4+ cytotoxic T cells, CD4+ helper T cells (e.g., Th1 or Th2 cells), CD4 / CD8 double positive T cells, tumor infiltrating T cells (TIL), thymocytes, memory T cells (e.g., central memory T cells, effector memory T cells, stem cell-like memory T cells, or stem cell memory T cells), and natural killer T cells, e.g., invariant natural killer T cells. In some embodiments, the T cells are CD39negCD69neg T cells or CD8+CD39negCD69neg cells, e.g., as described in Krishna et al., “Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer,” 2020 370(6522):1328-1334 (incorporated herein by reference in its entirety). Progenitor cells of the cellular immune system (e.g., progenitor cells of T lymphocytes) are also useful for presenting the TCRs disclosed herein, as these cells can differentiate, develop, or mature into effector cells. Thus, in certain embodiments, the mammalian cells are pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells), hematopoietic stem cells, or lymphoid progenitor cells. In certain embodiments, the hematopoietic stem cells or lymphoid progenitor cells are isolated and / or enriched, e.g., from bone marrow, umbilical cord blood, or peripheral blood. In some embodiments, the immune effector cells are CD4+ T cells. In some embodiments, the immune effector cells are CD8+ T cells. In one aspect, provided herein is a population of immune effector cells comprising a polycistronic vector as described herein.In some embodiments, the population of immune effector cells comprises CD4+ T cells and CD8+ T cells, hi some embodiments, the population of immune effector cells is in ex vivo culture.

[0617] 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 easily 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), sonication, 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 contents of which are incorporated herein by reference in their entirety (incorporated by reference in their entirety). In some embodiments, the polycistronic vector is introduced into the immune effector cell or population of immune effector cells via electroporation. Alternative delivery systems include 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 the cell, e.g., population of immune effector cells, ex vivo, in vitro, or in vivo. In some embodiments, the polycistronic vector is introduced into the cell, e.g., population of immune effector cells, ex vivo.

[0618] In some embodiments, co-expression of mbIL-15 with transgenic TCR in T cells produces a final drug product containing T stem cell memory cells (Tscm) capable of self-renewal and differentiation into other effector T cell subsets. Expression of mbIL-15 on T cells maintains a population of self-renewing T stem cell memory or T stem cell memory-like (Tscm-like) cells defined by the surface marker phenotypes CD45RA+CD45RO-CD62L+CD95+ or CD45RA+CD45RO+CD62L+CD95+, respectively. In some embodiments, expression of mbIL-15 on T cells can maintain Tscm or Tscm-like subsets as described above in the absence of external growth and survival factors (i.e., cytokines or antigenic stimulation).

[0619] In some embodiments, a population of T cells that co-express mbIL-15 with a transgenic TCR produced by a tricistronic vector described herein comprises greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% Tscm cells. In some embodiments, a population of T cells that co-express mbIL-15 with a transgenic TCR produced by a tricistronic vector described herein comprises greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% Tscm-like cells. In some embodiments, a population of T cells co-expressing mbIL-15 with a transgenic TCR produced by a tricistronic vector described herein comprises greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD45RA+CD45RO-CD62L+CD95+ cells. In some embodiments, a population of T cells co-expressing mbIL-15 with a transgenic TCR produced by a tricistronic vector described herein comprises greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD45RA+CD45RO+CD62L+CD95+ cells.

[0620] 5.8.1 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.

[0621] 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.

[0622] 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.).

[0623] In certain embodiments, the mammalian cells are a cell population that displays a TCR disclosed herein on the cell surface. The cell population may be heterogeneous or homogeneous. In certain embodiments, at least 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%) of the population are cells described herein. In certain embodiments, the population is substantially pure, and at least 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%) of the population is homogeneous. In certain embodiments, the population is heterogeneous and includes a mixed population of cells (e.g., the cells are of different cell types, developmental stages, origins, are isolated, purified, or enriched by different methods, are stimulated with different agents, and / or are manipulated by different methods). In certain embodiments, the cells are a population of peripheral blood mononuclear cells (PBMCs) (e.g., human PBMCs).

[0624] The cell population may be enriched or purified, as desired. In certain embodiments, regulatory T cells (e.g., CD25+ T cells) are depleted from the population, for example, by using an anti-CD25 antibody conjugated to a surface, such as a bead, particle, or cell. In certain embodiments, the anti-CD25 antibody is conjugated to a fluorescent dye (e.g., for use in fluorescence-activated cell sorting). In certain embodiments, cells expressing a checkpoint receptor (e.g., CTLA-4, PD-1, TIM-3, LAG-3, TIGIT, VISTA, BTLA, TIGIT, CD137, or CEACAM1) are depleted from the population, for example, by using an antibody that specifically binds to the checkpoint receptor conjugated to a surface, such as a bead, particle, or cell. In another embodiment, the T cell population can be selected to express one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-13, granzymes (e.g., granzyme B), and perforin, or other suitable molecules, e.g., other cytokines. For methods for determining such expression, see, e.g., PCT Publication No. 2013 / 126712, which is incorporated by reference in its entirety.

[0625] 5.8.2 Manufacturing method The engineered cells described herein can be produced by any method known in the art. Exemplary methods are set forth in U.S. Patent Publication No. 2020 / 0347350 and WO 2019 / 067242, which are incorporated by reference in their entireties.

[0626] 5.9 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).

[0627] 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.

[0628] 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.

[0629] 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.

[0630] Formulations to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0631] 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. 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.

[0632] 5.10 Kits In one aspect, provided herein is a kit comprising one or more of the pharmaceutical compositions, engineered immune effector cell (e.g., recombinant 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, for example, 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.

[0633] 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

[0634] 6. Working Example The presently disclosed embodiments are provided by way of illustration and description and are not intended to limit the scope of the present disclosure.

[0635] 6.1 Example 1: Construction of transposon plasmids To improve the uniformity and product manufacturability of multigene co-expression, recombinant nucleic acid SB transposon plasmids were constructed that contain polycistronic expression cassettes, each of which contains a transcriptional regulatory element operably linked to a polycistronic polynucleotide encoding the TCR alpha chain of TCR001 (referred to herein as "TCR alpha" or "A"), the TCR beta chain of TCR001 (referred to herein as "TCR beta" or "B"), and a membrane-bound IL-15 / IL-15Ralpha fusion protein (referred to herein as "mbIL15" or "15"), each separated by a furin recognition site and either a P2A element or a T2A element that mediates ribosome skipping to allow expression of separate polypeptide chains.

[0636] The TCR used in this example, TCR001, is a chimeric TCR with a mouse-derived constant region and human Vα and Vβ regions specific for the R175H mutation in the p53 protein (position 175 of the p53 protein is mutated from Arg to His) in the context of HLA-A*02:01.

[0637] Briefly, TCRα was generated with a human Vα region containing its N-terminal signal sequence (SEQ ID NO:1006) fused to a glutamic acid at position 2, with a mouse Cα region modified by substituting a cysteine ​​at amino acid position 48, a leucine at amino acid position 112, an isoleucine at amino acid position 114, and a valine at amino acid position 115 (SEQ ID NO:41). TCRβ was generated with a human Vβ region containing its N-terminal signal sequence (SEQ ID NO:2006) fused to an alanine at position 2, with a mouse Cβ modified by substituting a cysteine ​​at amino acid position 57 (SEQ ID NO:51). mbIL15 was constructed by linking human IL-15 (SEQ ID NO:76) to human IL-15Rα (SEQ ID NO:78) via a Gly-Ser rich linker peptide (SEQ ID NO:81), with an IgE signal sequence (SEQ ID NO:83) at the N-terminus of human IL-15. A schematic diagram of each of these three polypeptide constructs is shown in FIG. 1, from N-terminus (left) to C-terminus (right) for each construct.

[0638] To explore the effect of gene / element order on expression and function, eight tricistronic polynucleotide expression cassettes were generated with polynucleotides encoding each of TCRα, TCRβ, and mbIL15. In each expression cassette, these three elements were pairwise fused with a) a polynucleotide encoding a furin recognition site linked to a P2A element (SEQ ID NO:11) (referred to herein as "fP2A" or "P"), and b) a polynucleotide encoding a furin recognition site linked to a T2A element (SEQ ID NO:13) (referred to herein as "fT2A" or "T"). The resulting tricistronic expression cassettes containing suitable transcriptional regulatory elements were inserted between the ITRs of a Sleeping Beauty (SB) transposon plasmid. The 5' to 3' order of elements in the open reading frame (ORF) of each expression cassette and the SB plasmid is shown in Table E1, and a schematic of the ORFs of these eight expression cassettes is shown in Figure 2A.

[0639] [Table E1]

[0640] The polynucleotide sequences of the ORFs of these transposon plasmids are shown in Table E2.

[0641] [Table E2-1]

[0642] [Table E2-2]

[0643] [Table E2-3]

[0644]

Table E2-4

[0645]

Table E2-5

[0646]

Table E2-6

[0647]

Table E2-7

[0648]

Table E2-8

[0649]

Table E2-9

[0650]

Table E2-10

[0651]

Table E2-11

[0652]

Table E2-12

[0653]

Table E2-13

[0654] [Table E2-14]

[0655] The corresponding theoretical polypeptide translation products resulting from each ORF, without considering N-terminal signal sequence cleavage or ribosomal skipping at each P2A and T2A site, are shown in Table E3.

[0656] [Table E3-1]

[0657] [Table E3-2]

[0658] [Table E3-3]

[0659] [Table E3-4]

[0660] [Table E3-5]

[0661] For control purposes, three additional SB transposon plasmids were prepared. Plasmid 15 contains a monocistronic expression cassette, Cassette 15, encoding mbIL15. Plasmid APB contains a bicistronic expression cassette, Cassette APB, encoding TCR α (5') and TCR β (3') with an intervening fP2A element. Plasmid BPA contains a bicistronic expression cassette, Cassette BPA, encoding TCR β (5') and TCR α (3') with an intervening fP2A element. These expression cassettes, containing suitable transcriptional regulatory elements, were inserted between the ITRs of the SB transposon plasmids. The 5' to 3' order of elements in the ORFs of each control expression cassette and the SB plasmid is shown in Table E4, and a schematic of the ORFs of these three expression cassettes is shown in Figure 2B.

[0662] Table E4

[0663] A plasmid encoding the SB11 transposase, plasmid TA, was also constructed.

[0664] 6.2 Example 2: Generation and Evaluation of T Cells This example describes the generation and evaluation of T cells co-expressing TCRα, TCRβ, and mbIL15 from the plasmids described in Example 1. A schematic of the gene transfer process for both double transposition (using separate plasmids encoding TCRα / TCRβ and mbIL15) and single transposition (using a tricistronic plasmid encoding TCRα / TCRβ and mbIL15 together) is shown in Figure 3.

[0665] Briefly, peripheral blood mononuclear cells (PBMCs) were enriched from leukapheresis products obtained from normal donors (Discovery Life Sciences, Austin, TX). The resulting PBMCs were collected, cryopreserved, and stored in the vapor phase of a liquid nitrogen tank.

[0666] To generate TCR-T cells as described in this Example 2, the plasmids described in Example 1 were electroporated into enriched PBMCs. Briefly, cryopreserved PBMCs were thawed, resuspended in supplemented medium, and incubated in a 37°C / 5% CO2 incubator for 1 hour. The PBMC test articles listed in Table E5 were then prepared.

[0667] [Table E5]

[0668] The test articles were prepared as follows.

[0669] Group 1: Resting cells were harvested, spun down, resuspended in supplemented medium and incubated overnight in a 37° C. / 5% CO 2 incubator.

[0670] Groups 2-14: Resting cells were harvested, spun down, resuspended in electroporation buffer with the plasmids listed in Table E5, and electroporated. After electroporation, the cell suspension was collected, transferred to supplemented medium, and incubated overnight in a 37 °C / 5% CO2 incubator.

[0671] Within 24 hours after electroporation (day 1), cells were harvested from the culture, counted, and sampled by flow cytometry to determine mbIL15 and TCR transgene expression. Briefly, up to 1x106 cells of each test article were first stained with human Fc block (BD Biosciences 564220) to reduce background staining for 10 minutes at room temperature. The cell suspension was further stained with fluorochrome-conjugated antibodies (listed in Table 1) diluted in Brilliant Stain suspension (BD Biosciences 566349) for 30 minutes at 4°C. TCR expression was detected using PerCP-Cy5.5-conjugated anti-mouse TCRβ antibody specifically to the mouse constant region of TCRβ. Fluorescently conjugated antibodies used included CD3 (clone OKT-3), IL-15 (34559), CD8 (clone RPA-T8), and Invitrogen violet live / dead dye (Table E6).

[0672] [Table E6]

[0673] Cells were washed with FACS buffer (PBS, 2% FBS, 0.1% sodium azide). Data was acquired using a NovoCyte Quanteon flow cytometer system (Agilent) and analyzed with FlowJo software (version 10.7.1, TreeStar, Ashland, OR) to determine the percentage of each transgenic subpopulation (mbIL15+mTCR+, mbIL15negmTCR+, mbIL15+mTCRneg, mbIL15negmTCRneg) present in each test article. Unless otherwise stated, transgene expression was assessed for gated cell events, singlets, viable events, and CD3+ cells.

[0674] The flow cytometry results are shown in Figure 4 and Table E7.

[0675] [Table E7]

[0676] 6.3 Example 3: Generation and Evaluation of Expanded T Cells This example describes the generation and evaluation of T cells co-expressing TCRα, TCRβ, and mbIL15 from the plasmids described in Example 1. The TCR-T cells described in this Example 3 were generated similarly to those described in Example 2, except as noted below.

[0677] Briefly, cryopreserved PBMCs were thawed, resuspended in supplemented medium (IL-7+IL-15) and incubated for 1 hour in a 37° C. / 5% CO 2 incubator.

[0678] The test articles listed above in Table E5 were then prepared as follows.

[0679] Group 1: Resting cells were harvested, spun down, resuspended in recovery medium (50:50 medium containing IL-7+IL-15+n-acetylcysteine ​​(NAC)) and incubated overnight in a 37°C / 5% CO2 incubator.

[0680] Groups 2-14: Resting cells were harvested, spun down, resuspended in electroporation buffer with the plasmids listed in Table E5, and electroporated. After electroporation, the cell suspension was collected, transferred to recovery medium (50:50 medium containing IL-7+IL-15+NAC) and incubated overnight in a 37°C / 5% CO2 incubator.

[0681] Groups 3-14: Within 24 hours after electroporation (day 1), mTCR positive (mTCR+) cells were isolated using mTCR antibody and MACS® cell separation system (Miltenyi Biotec). Live cells from groups 1 and 2, as well as live TCR+ enriched cells from groups 3-14, were transferred to G-REX® culture plates (Wilson Wolf Manufacturing) and incubated with the first expansion medium (50:50 medium containing IL-21+IL-7) and irradiated allogeneic feeder cells and OKT3 antibody. Cells were fed periodically with cytokines. After 13 days of the first expansion phase, cells were harvested and mTCR and mbIL15 expression was detected on the CD3+ gated population with mouse TCR beta and IL-15 antibodies as described in Example 2. Cell counts and viability were measured with an NC3000 cell counter. Unless otherwise stated, transgene expression was assessed for gated cell events, singlets, viable events, and CD3+ cells.

[0682] Expression of mTCR and mbIL15, as well as cell viability, was assessed for each test article at two separate time points: 1) after electroporation (day 1), and 2) after the first expansion phase (day 13).

[0683] TCR expression after electroporation (day 1) is shown in Figures 5A-B. Figure 5A provides representative TCR expression data from each test article. Figure 5B provides TCR expression data from three donors presented as %mTCR+ cells among CD3+ cells.

[0684] TCR and mbIL15 expression after the first expansion phase (day 13) is shown in Figures 6A-C. Figure 6A provides representative TCR and mbIL15 expression data from each test article. Figure 6B provides TCR expression data from three donors presented as %mTCR+ cells from CD3+ cells, and Figure 6C provides %TCR+mbIL15+ cells from CD3+ cells.

[0685] TCR+ and TCR+mbIL15+ cell numbers were also assessed after the first expansion phase (day 13), as shown in Figures 7A-B. Figure 7A provides TCR expression data from three donors presented as total numbers of mTCR+ T cells, and Figure 7B provides total numbers of TCR+mbIL15+ T cells.

[0686] Cell viability after electroporation (day 1) and after the first expansion phase (day 13) is shown in Figures 8A and 8B, respectively.

[0687] Transgene expression and cell count data demonstrate that BP15TA and AP15TB are the most potent candidates with mbIL15+TCR+ T cells having the highest levels of TCR and mbIL15 expression. Viability data demonstrated that despite the size of the tricistronic mbIL15+TCR vector (groups 7-14), viability was similar to the two-vector co-transfection system (groups 5 and 6).

[0688] TCR-T cell functionality was also measured after the first expansion phase (day 13), as described below.

[0689] The activation of TCR-T cells generated by electroporation with different polycistronic plasmids was evaluated. After 13 days of the first expansion phase, cells were co-cultured with wild-type or mutant neoantigen peptide-pulsed T2 cells with endogenous expression of HLA-A*02:01. After overnight incubation, cells were harvested and induction of 4-1BB molecules on CD3+CD8+ cells was detected with 4-1BB antibody. The results are shown in Figure 9A-B and show that mbIL15 / TCR-T cells are highly avid and specific for the target neoantigen as measured by upregulation of 4-1BB costimulatory receptor with negligible recognition of the wild-type sequence. There was no significant difference in function between mbIL15 / TCR-T cells generated using different polycistronic plasmids.

[0690] The level of phosphorylated STAT5 was also assessed for TCR-T cells electroporated with the different polycistronic plasmids. After 13 days of the first expansion phase, cells were washed and incubated overnight in cytokine-free 50:50 medium to stabilize STAT5 phosphorylation. STAT5 phosphorylation was detected the next day on CD3+ cells using pSTAT5(pY694). The results are shown in Figure 10 and demonstrate that the expressed mbIL15 is functional. IL15 signaling was activated and STAT5 (downstream of the IL15 receptor) phosphorylation was induced. STAT5 phosphorylation in mbIL15 TCR-T cells generated with the different polycistronic plasmids was not significantly different between the plasmids, but was increased in all mbIL15 containing plasmids compared to the TCR-only condition lacking mbIL15.

[0691] Apoptosis levels 9 days after activation were evaluated for TCR-T cells electroporated with the different polycistronic plasmids. After 13 days of the first expansion phase, cells were washed and activated with CD3 / CD28 Dynabeads® (ThermoFisher) for 9 days. After activation, apoptosis of CD3+TCR+ cells was monitored with AnnexinV / 7ADD kit (Biolegend). The results are shown in FIG. 11 and show that expression of mbIL15 on CD3+TCR+ cells inhibited AICD (activation-induced cell death) as measured by fewer cells stained for AnnexinV and / or PI. This inhibition of AICD was not significantly different between the different polycistronic plasmids tested, nor did it differ from the two-vector system (APB+mbIL15 and BPA+mbIL15).

[0692] A second expansion phase was performed as described below, and vector copy number (VCN) after the second expansion phase was evaluated. Briefly, T cells from groups 3-14 were isolated by MACS using mTCR antibody. T cells from groups 2-14 were then incubated with second expansion medium (50:50 medium containing IL-21) and irradiated feeder cells and OKT3 antibody. Cells were fed periodically with cytokines. 15 days after the second expansion phase, cells were harvested and VCN was detected using qPCR as the average number of Sleeping Beauty transgene DNA copies per cell in the samples. Results are shown in Table E8, which shows that low levels of vector were detected in TCR-T cells and mbIL15 / TCR-T cells after two rounds of expansion.

[0693] [Table E8]

[0694] Conclusion: The set of data presented in this example indicates that BP15TA and AP15TB are the most potent candidates for generating mbIL15TCR-T cells with the highest levels of TCR and mbIL15 expression. All plasmids expressing mbIL15 were assessed for increased phosphorylation of STAT5, indicating mbIL15 expressed at sufficient levels to induce downstream signaling in T cells. Furthermore, co-expression of mbIL15 with transgenic TCR reduces AICD following T cell activation. Furthermore, all tricistronic mbIL15 / TCR plasmids tested yielded acceptable VCN values.

[0695] 6.4 Example 4: Evaluation of polycistronic TCR constructs with different mouse constant regions. This example evaluates the effect of different murine constant regions on the TCR constructs described in Examples 1-3.

[0696] Briefly, the amino acid sequences of the TCR α and TCR β chains discussed herein are identical to those described in Examples 1-3, except that the constant regions of each chain are not cysteine ​​substituted. Specifically, the TCR α chain was generated by fusing the human Vα region, including its N-terminal signal sequence (SEQ ID NO: 1006), with glutamic acid at position 2 to a mouse Cα region modified by substituting leucine at amino acid position 112, isoleucine at amino acid position 114, and valine at amino acid position 115 (SEQ ID NO: 42). The TCR β chain was generated by fusing the human Vβ region, including its N-terminal signal sequence (SEQ ID NO: 2006), with alanine at position 2 to a mouse wild-type Cβ (SEQ ID NO: 52). The constructs containing the cysteine ​​substituted constant domains as described in Examples 1-3 are referred to below as "S versions" and the newly generated constructs containing the non-cysteine ​​substituted constant domains are referred to below as "N versions". A schematic of these constructs is provided in FIG.

[0697] The unified plasmid "NU version" referred to below differs in the nucleotide sequence of the TCR constant region compared to the "N version". All "NU versions" contain the same nucleotide sequence encoding the TCR constant region (Cα and Cβ), but the amino acid sequence of the TCR constant region encoded by the "NU version" is identical to that of the "N version". There are no other differences between the "N version" and the "NU version".

[0698] To generate the TCR-T cells described in this Example 4, the plasmids described above were electroporated into enriched PBMCs. Briefly, cryopreserved PBMCs were thawed, replaced with 50:50 medium, and electroporated. The PBMC test articles listed in Table E9 were then prepared. Where cells are indicated to be transposed, they were co-electroporated with the indicated plasmid and plasmid TA.

[0699] [Table E9]

[0700] The test articles were prepared as follows.

[0701] Group 2.1: Cells were harvested, spun down, resuspended in recovery medium (50:50 medium containing IL-7+IL-15+n-acetylcysteine ​​(NAC)) and incubated overnight in a 37°C / 5% CO2 incubator.

[0702] Groups 2.2-2.9: Cells were harvested, spun down, resuspended in electroporation buffer with the plasmids listed in Table E9, and electroporated. After electroporation, the cell suspension was collected, transferred to recovery medium (50:50 medium containing IL-7+IL-15+NAC) and incubated overnight in a 37°C / 5% CO2 incubator.

[0703] Within 24 hours after electroporation (day 1), live cells were transferred to G-REX® culture plates (Wilson Wolf Manufacturing) and incubated with the first expansion medium (50:50 medium containing IL-21+IL-7+IL-12+T cell TransAct™). Cells were fed periodically with cytokines. After 11 days of the first expansion phase, TCR+ cells were isolated with mTCR antibody. The isolated TCR+ T cells were transferred to G-REX® culture plates (Wilson Wolf Manufacturing) and incubated with the second expansion medium (50:50 medium containing 3000 IU / ml of IL-2+T cell TransAct™). Cells were fed periodically with cytokines. After 11 or 16 days of the second expansion phase, cells were harvested and subjected to various assays described below.

[0704] Transgene expression was evaluated for T cells electroporated with the different polycistronic plasmids. Cells were harvested on day 1 (after electroporation), before enrichment on day 11 (after the first expansion phase), after enrichment on day 11, and on day 22 (after the second expansion phase) and expression of mTCR and mbIL15 was detected on the CD3+ gated population with mouse TCR beta and IL-15Rα antibodies. On day 1 after electroporation, TCR expression levels were similar between the different versions of the polycistronic plasmid (Figure 13A). Before enrichment on day 11, TCR expression in mbIL15 / TCRT cells was lower compared to TCR alone, whereas TCR expression was comparable after enrichment on day 11 (Figure 13B). On day 22, when cells were harvested, TCR expression was comparable with no notable differences between the "S" and "N" versions of the polycistronic plasmid. Co-expression of TCR and mbIL15 was found to be similar between the different versions of the polycistronic plasmid throughout the process (Figures 14-15).

[0705] The expansion rates of total cell numbers (Figures 16A-B) and mTCR+ cell numbers (Figures 17A-B) were assessed for T cells electroporated with the different polycistronic plasmids. The expansion rate values ​​were calculated as follows: cell number at day 11 / cell number at day 1 (Figure 16A), and cell number at day 22 / cell number at day 11 (Figure 16B). Cells transposed with MbIL15 / TCR tricistronic plasmids tended to expand less than cells transposed with TCR-only bicistronic plasmids during both the first and second expansion phases. However, a significant degree of expansion was achieved in all groups, and no differences were seen between the different versions of the polycistronic plasmids. The mTCR+ cell numbers were calculated as follows: total cell number x CD3 population (%) x mTCR population (%).

[0706] The transgene expression and cell growth data presented above indicate that cells generated using the N and NU versions of the polycistronic plasmid were not phenotypically different from cells generated using the S version of the polycistronic plasmid.

[0707] The second expansion phase was extended to 16 days (by logistic load) to perform pSTAT5 assay, 4-1BB induction assay, and IFN-γ assay. Phosphorylation of STAT5 in T cells on day 27 was detected on CD3+ cells using pSTAT5(pY694). The pSTAT5 data shown in FIG. 18 indicated that the expressed mbIL15 was functional. It activated IL15 signaling and induced phosphorylation of STAT5 (downstream of the IL15 receptor). Phosphorylation of STAT5 in mbIL15 TCR-T cells generated with different versions of the polycistronic plasmid was not significantly different. The level of phosphorylated STAT5 tended to be higher in cells co-expressing mbIL15 and TCR compared to cells expressing TCR alone.

[0708] To assess antigen-specific activation of generated TCR-T cells, overnight co-culture of generated TCR-T cells with wild-type or mutant neoantigen-pulsed DCs (HLA-matched) was performed after a second expansion phase of 16 days, and 4-1BB induction and IFN-γ secretion were measured. Induction of 4-1BB on CD3+CD8+ cells was detected with 4-1BB antibody. Secretion of IFN-γ was measured by ELISA (clones 2G1 and B133.5, Thermo Fisher). The 4-1BB induction results shown in Figure 19A and IFN-γ secretion results shown in Figure 19B do not indicate that the functions of mbIL15 TCR-T cells generated with different versions of the polycistronic plasmid are significantly different.

[0709] Long-term withdrawal (LTWD) assays were performed to examine transgene expression, survival, and activation of T cells cultured under cytokine-free conditions. LTWD assays were performed as follows: Day 22 engineered T cells (after the first and second expansion phases) were transferred into T25 flasks and cultured in cytokine-free medium (50:50) for 4 weeks. 50% of the medium was replaced weekly. For control groups (groups 2.2 and 2.3, Table E9), cells were treated with 300 U / ml IL-2 twice a week with 50% of the medium replaced. Flow data were acquired using a NovoCyte Quanteon flow cytometer system (Agilent) and analyzed with FlowJo software (version 10.7.1, TreeStar, Ashland, OR). (Data n=4, pooled from two independent experiments)

[0710] After 4 weeks of LTWD incubation, expression of mTCR was detected in the CD3+ gated population with a mouse TCR beta antibody (Figure 20A) and cell counts and viability were accessed (Figure 20B). This mTCR expression and cell count data showed no significant differences between mbIL15 TCR-T cells generated with different versions of the polycistronic plasmid. The number of viable cells decreased after long-term cytokine withdrawal in all groups, but cells from the group co-expressing mbIL15 and TCR survived 5-6 times more compared to cells from the TCR-only group.

[0711] Activation of TCR-T cells after LTWD culture was assessed by 4-1BB induction and IFN-γ secretion after overnight coculture with wild-type or mutant neoantigen (10 μg / mL)-pulsed DCs (HLA-matched). Induction of 4-1BB in CD8+ T cells was detected with 4-1BB antibody (Figures 21A-B) and IFN-γ secretion was measured by ELISA (Figures 22A-B) as described above. These data indicate that mbIL15 TCR-T cells surviving from LTWD cultures retained their specific functions and showed stronger activation than T cells from the control TCR-only group (IL-2 treatment), whereas the functions of mbIL15 TCR-T cells generated with different versions of the polycistronic plasmid were not significantly different.

[0712] We also evaluated the memory phenotype of TCR-T cells electroporated with different polycistronic vectors. T cell memory subsets are defined as follows: CD45RA+CD45RO+CD62L+CD95+=stem cell memory-like (Tscm-like), CD45RA+CD45RO-CD62L+CD95+=stem cell memory (Tscm), CD45RA-CD45RO+CD62L+CD95+=central memory (Tcm), CD45RA-CD45RO+CD62L-CD95+=effector memory (Tem). T cell effector (Teff) is defined as CD45RA+CD45RO+CD62L-CD95+. The pie charts in Figures 23A-C show the mean frequencies of live CD3+ T cell memory and effector subsets in cells transfected with the test plasmids at day 11 after the first expansion phase (Figure 23A), day 22 after the second expansion phase (Figure 23B), and after 4 weeks of LTWD culture (Figure 23C).

[0713] Memory phenotype data show the kinetics of TCR-T memory and effector differentiation. At days 11 and 22 after expansion, there is no difference between the different polycistronic TCR plasmids (Figures 23A-B). Compared to TCR-T cells without mbIL15, TCR-T cells expressing mbIL15 had more Tscm and Teff cells and fewer Tcm cells. After 4 weeks of culture in the absence or presence of IL-2, TCR-T cells differentiated predominantly into Teff cells (>85%). TCR-T cells expressing mbIL15 were cultured for 4 weeks in the absence of cytokines differentiated into three major subsets: Teff, Tscm-like and Tscm cells (Figure 23C). These results suggest that mbIL15 is sufficient to support the Tscm phenotype.

[0714] Conclusion: mbIL15 TCR-T cells generated from different versions of the polycistronic plasmid showed comparable characteristics including TCR expression, memory phenotype, specificity, and IFN-γ secretion. This data supports that the removal of cysteine ​​substitutions in the mouse constant domains used in the first generation vectors and the use of a unified mouse constant region do not result in any significant changes in the mbIL15 TCR-T cell product.

[0715] 6.5 Example 5: Generation and evaluation of T cells generated using various tricistronic TCR / mbIL15 vectors This example describes the evaluation of T cells expressing mbIL15 in combination with different TCR alpha / beta chains generated using a tricistronic vector described below. Similar to the vector described in Example 4, the tricistronic expression cassettes used in this example each contain a transcriptional regulatory element operably linked to a polycistronic polynucleotide encoding a TCR alpha chain (referred to herein as "TCR alpha" or "A"), a TCR beta chain (referred to herein as "TCR beta" or "B"), and a membrane-bound IL-15 / IL-15Ralpha fusion protein (referred to herein as "mbIL15" or "15"), each separated by a furin recognition site and either a P2A element or a T2A element that mediates ribosomal skipping to allow expression of separate polypeptide chains.

[0716] The nine TCRs used in this example are each directed against a different target, as shown in Table E10. The Vα and Vβ amino acid sequences for each of the nine TCRs listed correspond to the sequences provided in Table 6. Each TCR α chain was generated by fusing the Vα sequence to a mouse Cα region modified by substituting leucine at amino acid position 112, isoleucine at amino acid position 114, and valine at amino acid position 115 (SEQ ID NO: 42). Each TCR β chain was generated by fusing the Vβ sequence to a mouse wild-type Cβ (SEQ ID NO: 52).

[0717] [Table E10]

[0718] For each of the above TCRs, three vectors were constructed and evaluated: 1) TCR only (BA), 2) A15B, and 3) B15A. The TCR only (BA) vector contains a bicistronic expression cassette encoding the TCR β and TCR α chains in the following 5' to 3' orientation: TCR β-TCR α, separated by a furin recognition site and a P2A element. The AP15TB vector contains a tricistronic expression cassette encoding the TCR α chain, TCR β chain, and mbIL15 in the following 5' to 3' orientation: TCR α-mbIL15-TCR β. The BP15TA vector contains a tricistronic expression cassette encoding the TCR α chain, TCR β chain, and mbIL15 in the following 5' to 3' orientation: TCR β-mbIL15-TCR α.

[0719] The TCR-T cells described in this example were generated similarly to those described in Examples 2-4, except as indicated below: Where cells are indicated to have been transposed, unless otherwise stated, the cells were co-electroporated with the indicated plasmid as well as plasmid TA or a similar transposase-expressing plasmid.

[0720] Briefly, cryopreserved PBMCs were thawed, resuspended in 50:50 medium and placed in a 37° C. / 5% CO 2 incubator prior to electroporation.

[0721] The test articles listed in Table E11 were then prepared.

[0722] [Table E11]

[0723] Test articles were prepared in three batches as follows (Batch 1 = Groups 3.1-3.13, Batch 2 = Groups 3.14-3.26, Batch 3 = Groups 3.27-3.30).

[0724] Groups 3.1, 3.14, and 3.27: Cells were harvested, spun down, resuspended in recovery medium (50:50 medium containing IL-7+IL-15+n-acetylcysteine ​​(NAC)) and incubated overnight in a 37°C / 5% CO2 incubator.

[0725] Groups 3.2-3.13, 3.15-3.26, and 3.28-3.30: Cells were harvested, spun down, resuspended in electroporation buffer with the plasmids listed in Table E10, and electroporated. After electroporation, the cell suspension was collected, transferred to recovery medium (50:50 medium containing IL-7+IL-15+NAC) and incubated overnight in a 37°C / 5% CO2 incubator.

[0726] Within 24 hours after electroporation (day 1), live cells were transferred to G-REX® culture plates (Wilson Wolf Manufacturing) and incubated with the first expansion medium (50:50 medium containing IL-21+IL-7+IL-12+T cell TransAct™). Cells were fed periodically with cytokines. After 11 days of the first expansion phase, TCR+ cells were isolated with mTCR antibodies. The isolated TCR+ T cells were transferred to G-REX® culture plates (Wilson Wolf Manufacturing) and incubated with the second expansion medium (50:50 medium containing 3000U / ml IL-2+T cell TransAct™). During this second expansion phase, cells were fed periodically with cytokines. After 11 or 16 days of the second expansion phase, cells were harvested and subjected to various assays described below.

[0727] Transgene expression was evaluated for T cells electroporated with the different polycistronic plasmids. Cells were harvested on day 1 (after electroporation), day 11 (after the first expansion phase), and day 22 (after the second expansion phase) and expression of mTCR and mbIL15 was detected on the CD3+ gated population with mouse TCR beta and IL-15Rα antibodies. Results are shown in Figures 24-28.

[0728] The expansion rates of total cell number and mTCR+ cell number were evaluated for T cells electroporated with different polycistronic plasmids. Expansion rate values ​​were calculated as follows: cell number on day 11 / cell number on day 1 and cell number on day 22 / cell number on day 11. mTCR+ cell number was calculated as follows: total cell number x CD3 population (%) x mTCR population (%). The results are shown in Table E12.

[0729] [Table E12]

[0730] Cells generated using polycistronic plasmids containing different TCR sequences were not phenotypically distinct from each other, as demonstrated by transgene expression and cell proliferation data.

[0731] To assess activation of generated TCR-T cells, overnight co-culture of generated TCR-T cells with wild-type or mutant neoantigen-pulsed DCs (HLA-matched) was performed after a second expansion phase of 16 days and 4-1BB induction and IFN-γ secretion were measured. Induction of 4-1BB on CD3+CD8+ cells was detected with 4-1BB antibody. IFN-γ secretion measured with ELISA antibody pair. 4-1BB induction results are shown in Figures 29A-I and IFN-γ secretion results are shown in Figures 30A-I. The results show that mbIL-15TCR-T cells were highly avid and specific for the target neoantigens when challenged with their cognate neoantigens as measured by upregulation of 4-1BB costimulatory receptors and secretion of IFN-γ with negligible recognition of the wild-type sequence.

[0732] All data from electroporation of the TCR bet to the second expansion phase demonstrated that the tricistronic system expressing TCRα, TCRβ and mbIL15 in one plasmid successfully generated mbIL15 TCR-T cells, and the characteristics of the generated mbIL15 TCR-T cells were comparable to TCR-T cells in terms of transgene expression, cell proliferation, and functional specificity (4-1BB induction and IFN-γ secretion).

[0733] Cytolytic activity of TCR-T cells was assessed for T cells electroporated with a polycistronic plasmid encoding TCR001+ / -mbIL15 generated as described above (overnight recovery + 11-day first expansion + 11-day second expansion), then harvested and frozen on day 22. On the day of the experiment, frozen day 22 TCR-T cells were thawed and allowed to recover for 3 days in medium containing 3000 U / ml IL-2. The recovered TCR-T cells were then incubated with AU565 (Mut+HLAneg) or Tyk-nu (Mut+HLA+) cells. After overnight incubation, the remaining T cells were washed extensively and the extent of viable cells left in the culture after TCR-specific cytolysis was measured using a CellTiter Glo luminescence-based assay. The results are shown in Figure 31.

[0734] Specific lysis was calculated from background subtracted values ​​as follows:

[0735]

number

[0736] Cytolytic activity of TCR-T cells was also evaluated for T cells electroporated with polycistronic plasmids encoding TCR022+ / -mbIL15 or TCR075+ / -mbIL15 generated as described above (overnight recovery + 11 days first expansion + 11 days expansion) and then harvested and frozen on day 22. On the day of the experiment, frozen day 22 TCR-T cells were thawed and recovered for 3 days in medium containing 3000U / ml IL-2. Meanwhile, Saos-2 cells were plated in 96-well plates. After overnight incubation, HLA*11:01 plasmid was transfected into Saos-2 cells and the next day, WT or MUT neoantigenic peptides (1ug / ml) were loaded into the transfected Saos-2 cells for 2 hours. The recovered TCR-T cells were then incubated overnight with the resulting Saos-2 cells. After overnight incubation, the remaining T cells were washed extensively and the extent of viable cells left in the cultures after TCR-specific cell lysis was measured using the CellTiter Glo luminescence-based assay, and the results are shown in Figures 32A-B.

[0737] Specific lysis was calculated from background subtracted values ​​as follows:

[0738]

number

[0739] Long-term withdrawal (LTWD) assays were performed to examine transgene expression, survival, and activation of T cells cultured under cytokine-free conditions. LTWD assays were performed as follows: Day 22 engineered T cells (after the first and second expansion phases) were transferred into T25 flasks and cultured in cytokine-free medium (50:50) for 4 weeks. 50% of the medium was replaced weekly. For the control TCR only (BA) group, cells were treated with 300 U / ml IL-2 twice a week with 50% of the medium replaced. Flow data were acquired using a NovoCyte Quanteon flow cytometer system (Agilent) and analyzed with FlowJo software (version 10.7.1, TreeStar, Ashland, OR). (Data n=4, pooled from two independent experiments)

[0740] After 4 weeks of LTWD incubation, expression of mTCR was detected in the CD3+ gated population with a mouse TCR beta antibody (Figure 33), and cell numbers and viability were accessed (Figures 34A-C). The mTCR expression and cell number data showed no significant differences between mbIL15 TCR-T cells generated with different polycistronic plasmids. The number of viable cells decreased after long-term cytokine withdrawal in all groups, but cells from the group co-expressing mbIL15 and TCR survived 5-6 times more compared to cells from the TCR-only group.

[0741] Activation of TCR-T cells after LTWD culture was assessed by 4-1BB induction and IFN-γ secretion after overnight coculture with wild-type or mutant neoantigen-pulsed DCs (HLA-matched). As described above, induction of 4-1BB in CD3+CD8+ cells was detected with 4-1BB antibody (Figure 35A-I), and IFN-γ secretion was measured using an ELISA antibody pair (Figure 36A-C). Comparison of 4-1BB induction assessed for T cells harvested on day 27 and after LTWD is shown in Figure 37A-I. These data indicate that mbIL15 TCR-T cells surviving from LTWD cultures were still functional and more strongly activated than cells from the TCR-only group. The data also show that 4 weeks after cytokine withdrawal (LTWD), mbIL15 TCR-T cells showed even stronger induction of 4-1BB compared to those cells after the second expansion step.

[0742] We also evaluated the memory phenotype of TCR-T cells electroporated with different polycistronic vectors. T cell memory subsets are defined as follows: CD45RA+CD45RO+CD62L+CD95+=stem cell memory-like (Tscm-like), CD45RA+CD45RO-CD62L+CD95+=stem cell memory (Tscm), CD45RA-CD45RO+CD62L+CD95+=central memory (Tcm), CD45RA-CD45RO+CD62L-CD95+=effector memory (Tem). T cell effector (Teff) is defined as CD45RA+CD45RO+CD62L-CD95+. The data in Tables E13 and E14 and representative pie charts in Figures 38-40 show the mean frequencies of live CD3+ T cell memory and effector subsets in cells transfected with the test plasmids at day 11 post-expansion (Table E13 and Figure 38), day 22 post-expansion (Table E14 and Figure 39), and after 4 weeks of LTWD culture (Figures 40A-E).

[0743] [Table E13]

[0744] [Table E14]

[0745] The memory phenotype data show the kinetics of TCR-T memory and effector differentiation. Addition of mbIL15 to TCR-T cells resulted in a change in the memory phenotype in the expansion product to contain fewer central memory cells (Tcm) and more effector (Teff) and stem cell memory (Tscm) populations compared to conventional TCR-T cells. After 4 weeks of culture in the presence of IL-2, TCR-T cells differentiated primarily into Teff cells. TCR-T cells expressing mbIL15 were cultured for 4 weeks in the absence of cytokines differentiated into three major subsets; Teff, Tscm-like and Tscm cells. These results suggest that mbIL15 is sufficient to direct T cell differentiation toward the Tscm phenotype.

[0746] Conclusion: mbIL15 TCR-T cells were successfully generated using 18 different constructs (2 different orientations, AP15TB and BP15TA x 9 TCR). Addition of mbIL15 to TCR-T cells resulted in a change in the memory phenotype in the expansion products to contain fewer central memory cells (Tcm) and more effector (Teff) and stem cell memory (Tscm) populations compared to conventional TCR-T cells. Furthermore, long-term withdrawal of cytokine support (LTWD) showed a survival of a fraction of mbIL15 TCR-T cells that was significantly higher than the survival of TCR-T cells lacking mbIL15. Functional and phenotypic evaluation of persistent mbIL15 TCR-T cells revealed that they retained functional neoantigen specificity and potency, while showing a predominance of TscmTCR-T cells that could regenerate the TCR-T cell effector pool. This suggested that mbIL15 TCR-T cells are likely to establish long-lived tumor-specific TCR-T cells that may overcome suppression by the tumor microenvironment or other negative regulators.This preclinical data supports the clinical application of the mbIL15 TCR-T cell platform and provides evidence that this strategy may result in improved efficacy of cancer treatment.

[0747] 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.

[0748] 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. Other embodiments are within the scope of the following claims.

Claims

1. 1. A recombinant vector comprising a polycistronic expression cassette, the polycistronic expression cassette comprising a transcriptional regulatory element operably linked to a polycistronic polynucleotide, the polycistronic polynucleotide comprising: a. a first polynucleotide sequence encoding a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region; b. a second polynucleotide sequence comprising a first 2A element; and c. a third polynucleotide sequence encoding a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ) region; d. a fourth polynucleotide sequence comprising a second 2A element; and e. a fifth 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.

2. 2. The recombinant vector of claim 1, wherein either or both of the first 2A element and the second 2A element are independently a P2A element, a T2A element, an F2A element, or an E2A element.

3. 3. The recombinant vector of claim 2, wherein the P2A element (a) comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 or 20, or the amino acid sequence of SEQ ID NO: 18 or 20 containing one, two, or three amino acid modifications, or (b) 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: 19 or 21.

4. 3. The recombinant vector of claim 2, wherein the T2A element (a) comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:22 or 24, or the amino acid sequence of SEQ ID NO:22 or 24 containing one, two, or three amino acid modifications; or (b) 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:23 or 25.

5. 5. The recombinant vector of claim 1, wherein either or both of the second polynucleotide sequence and the fourth polynucleotide sequence independently encode a furin recognition site, and optionally the furin recognition site comprises: (a) the amino acid sequence of SEQ ID NO: 2 or 4, or the amino acid sequence of SEQ ID NO: 2 or 4 with one, two, or three amino acid modifications; or (b) the amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 3 or 5, or the polynucleotide sequence of SEQ ID NO: 3 or 5 with one, two, or three nucleotide modifications.

6. 6. The recombinant vector of any one of claims 1 to 5, wherein the second polynucleotide sequence (a) comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of SEQ ID NO: 10 containing one, two, or three amino acid modifications; or (b) 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:

11.

7. 7. The recombinant vector of any one of claims 1 to 6, wherein the fourth polynucleotide sequence (a) comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 containing one, two, or three amino acid modifications; or (b) 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:

13.

8. 8. The recombinant vector of any one of claims 1 to 7, wherein the IL-15, or a functional fragment or variant thereof, comprises: (a) an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:76; or (b) an amino acid sequence 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:

77.

9. 9. The recombinant vector of any one of claims 1 to 8, wherein the IL-15Rα, or a functional fragment or variant thereof, comprises: (a) an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:78; or (b) an amino acid sequence 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:

79.

10. The recombinant vector of any one of claims 1 to 9, wherein the Cα region comprises: (a) an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 40 to 49; or (b) an amino acid sequence encoded by a 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: 55, 57, or 58.

11. The recombinant vector of any one of claims 1 to 10, wherein the Cβ region comprises: (a) an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:50-54 or 60; or (b) an amino acid sequence 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 or 59.

12. The polycistronic polynucleotide is arranged, in order from 5' to 3', as follows: (a) the first polynucleotide sequence, the second polynucleotide sequence, the third polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence; (b) the first polynucleotide sequence, the fourth polynucleotide sequence, the third polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence; (c) the first polynucleotide sequence, the second polynucleotide sequence, the fifth polynucleotide sequence, the fourth polynucleotide sequence, and the third polynucleotide sequence; (d) the first polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, the second polynucleotide sequence, and the third polynucleotide sequence; (e) the third polynucleotide sequence, the second polynucleotide sequence, the first polynucleotide sequence, the fourth polynucleotide sequence, and the fifth polynucleotide sequence; (f) (g) the third polynucleotide sequence, the fourth polynucleotide sequence, the first polynucleotide sequence, the second polynucleotide sequence, and the fifth polynucleotide sequence, (h) the third polynucleotide sequence, the fourth polynucleotide sequence, the fifth polynucleotide sequence, the second polynucleotide sequence, and the first polynucleotide sequence, (i) the fifth polynucleotide sequence, the second polynucleotide sequence, the first polynucleotide sequence, the fourth polynucleotide sequence, and the third polynucleotide sequence, (j) the fifth polynucleotide sequence, the fourth polynucleotide sequence, the first polynucleotide sequence, the second polynucleotide sequence, and the third polynucleotide sequence, (k) the fifth polynucleotide sequence, the second polynucleotide sequence, the third polynucleotide sequence, the fourth polynucleotide sequence, and the first polynucleotide sequence, or (l)The recombinant vector according to any one of claims 1 to 11, comprising the fifth polynucleotide sequence, the fourth polynucleotide sequence, the third polynucleotide sequence, the second polynucleotide sequence, and the first polynucleotide sequence.

13. The polycistronic polynucleotide comprises: (a) a sixth polynucleotide sequence comprising a third 2A element, and The recombinant vector of any one of claims 1 to 12, further comprising (b) a seventh polynucleotide sequence comprising a marker protein.

14. The Vα region (a) comprises complementarity determining region 1 α (CDR1α), CDR2α, and CDR3α comprising the amino acid sequences of SEQ ID NOs: 1001+10n, 1002+10n, and 1003+10n, respectively, where n is an integer between 0 and 79; (b) comprises the CDR1α, CDR2α, and CDR3α from a Vα region comprising the amino acid sequence of SEQ ID NO: 1004+10n, 1005+10n, 1006+10n, or 1007+10n, where n is an integer between 0 and 79; or (c)or (d) an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1004+10n, 1005+10n, 1006+10n, or 1007+10n, where n is an integer from 0 to 79; or (e) an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1004+10n, where the Vβ region is or wherein n is an integer from 0 to 79; or wherein the Vα region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1005+10n; and wherein the Vβ region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2005+10n. or the Vα region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1006+10n, and the Vβ region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2006+10n, and n is an integer from 0 to 79.

14. The recombinant vector of any one of claims 1 to 13, wherein the Vα region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1007+10n, and the Vβ region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2007+10n, where n is an integer from 0 to 79.

15. The Vβ region (a) comprises CDR1β, CDR2β, and CDR3β comprising the amino acid sequences of SEQ ID NOs: 2001+10n, 2002+10n, and 2003+10n, respectively, where n is an integer between 0 and 79; (b) comprises the CDR1β, CDR2β, and CDR3β from a Vβ region comprising the amino acid sequence of SEQ ID NO: 2004+10n, 2005+10n, 2006+10n, or 2007+10n, where n is an integer between 0 and 79; or (c) The recombinant vector of any one of claims 1 to 14, comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2004+10n, 2005+10n, 2006+10n, or 2007+10n, wherein n is an integer from 0 to 79.

16. 16. The recombinant vector of any one of claims 1 to 15, wherein the TCR alpha chain (a) comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1008+10n, where n is an integer between 0 and 79; (b) comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1009+10n, where n is an integer between 0 and 79; or (c) comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1010+10n, where n is an integer between 0 and 79.

17. The recombinant vector of any one of claims 1 to 16, wherein the TCR beta chain (a) comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2008+10n, where n is an integer between 0 and 79; (b) comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2009+10n, where n is an integer between 0 and 79; or (c) comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2010+10n, where n is an integer between 0 and 79.

18. A polynucleotide encoding an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 163, 164, 165, 167, 169, 170, and 171; or comprising a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 232, 235, 236, 238, 239, 241, 242, and 243.

19. a. a first cistron comprising a 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; b. a second cistron comprising a polynucleotide sequence encoding a TCR beta chain comprising a Vβ region and a Cβ region; and c. a third cistron comprising a polynucleotide sequence encoding a TCR alpha chain comprising a Vα region and a Cα region, The cell population is a T cell population that comprises more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% CD45RA+CD45RO-CD62L+CD95+ cells.

20. A therapeutic agent for cancer, an autoimmune disease, or an autoimmune disorder, comprising a recombinant vector according to any one of claims 1 to 17, or a cell population comprising the polynucleotide according to claim 18, or a cell population according to claim 19.

Images