Expression of novel cell tags

Abstract

To provide a polypeptide construct and polynucleotide that can be expressed intracellularly for controlling safety issues in the administration of modified immune cells into tumor-bearing individuals in cell therapies, for instance side effects such as toxicity, tumor lysis and cytokine release syndrome in the case of CAR-T cell therapy.SOLUTION: Provided is a polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of at least a HER1 domain III or a fragment thereof, and a truncated HER1 domain IV, the HER1 polypeptide binding an anti-HER1 antibody, and the HER1 polypeptide being expressed in an engineered cell.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 516,639, filed on June 7, 2017, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 30, 2018, is named 50471_708_601_SL.txt and is 464,445 bytes in size.

Background Art

[0003] Cell therapy is promising for treating diseases that cannot be adequately treated by conventional pharmaceuticals. Blood transfusion was the first type of cell therapy for treating hematological malignancies. Recent advances in cell isolation techniques, induction techniques, and gene transfer techniques have enabled the genetic modification of various cell types (primary cells and immortalized cells) for the treatment of diverse diseases, such as cancer, cardiovascular diseases, skin diseases, neurological diseases, and ophthalmic diseases. In many cases, it is extremely important to enrich the genetically modified cell therapy product and / or eliminate non - genetically modified cell types or other cell types to achieve the purity necessary to enable the expansion of the selected cells for therapeutic purposes. Additionally, adoptive cell immunotherapy using, for example, cytokines, chimeric antigen receptors (CARs), and T - cell receptors (TCRs) has been shown to be extremely promising for directing the killing of tumor cells. This novel ​​​​​​The technology is promising, but the administration of modified immune cells to individuals bearing tumors poses safety issues , for example, in the case of CAR-T cell therapy, toxicity, tumor lysis, and cytokine release syndrome (i.e., "cytokine storm") have not been absent. In order to fully utilize the therapeutic potential brought about by adoptive T cell immunotherapy, it is essential to control side effects such as cytokine storm during treatment.

[0004] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

[0005] Polypeptide constructs and polynucleotides capable of being expressed intracellularly are provided to address one or more of the above deficiencies.

[0006] Polypeptide constructs and polynucleotides encoding the polypeptide constructs, and polypeptide constructs comprising truncated variants of native polypeptides are provided. In some embodiments, the polypeptide construct may further comprise a transmembrane domain or a fragment thereof, a signal peptide, and / or a peptide linker. Also presented herein are engineered cells expressing the polynucleotide and the polypeptide construct. The engineered cell expresses the polypeptide construct on the cell surface, thereby, in some embodiments, providing a cell marker (or "cell tag") that uniquely identifies the engineered cell. ​

[0007] Further provided herein are polypeptide constructs and polynucleotides encoding the polypeptide constructs, polypeptides comprising a truncated variant of a native polypeptide and a transmembrane domain. In certain embodiments, the truncated variant comprises an extracellular domain or a portion thereof and a transmembrane domain or a portion thereof. In some embodiments, the polypeptide construct can comprise a transmembrane domain from a different native protein and a truncated variant. Optionally, the transmembrane domain that is a truncated variant of the polypeptide construct is a single-pass transmembrane domain. In other cases, the transmembrane domain of the polypeptide construct is a multi-pass transmembrane domain.

[0008] Also provided herein are polypeptide constructs and polynucleotides encoding the polypeptide constructs, polypeptide constructs comprising a transmembrane dimerization domain capable of coupling a cell surface polypeptide (e.g., a truncated variant fused to a transmembrane domain) to a second cell surface polypeptide at the cell surface. In certain embodiments, coupling a cell surface polypeptide via the transmembrane dimerization domain can amplify a signal derived from the cell surface polypeptide as compared to a non-dimerized conformation.

[0009] Still further provided herein are polypeptide constructs and polynucleotides encoding the polypeptide constructs, polypeptide constructs comprising domains or fragments thereof from different native proteins. In some embodiments, the polypeptides described herein The construct may include a truncated variant of a native polypeptide, a transmembrane domain, a truncated variant, an optional peptide linker that connects the truncated variant to the transmembrane domain, and a signal peptide that directs the polypeptide construct to the cell surface. For example, in some embodiments, the polypeptide construct contains a truncated variant or a fragment thereof that is directly or indirectly fused to a transmembrane domain or a fragment thereof and is derived from a native protein different from the transmembrane domain or its fragment. In some embodiments, a particular domain of the polypeptide construct described herein (e.g., the extracellular domain) is chimeric and contains amino acid sequences derived from different native proteins.

[0010] In some cases, provided are methods and compositions that include a polypeptide construct comprising a cell surface polypeptide and a transmembrane dimerization domain, wherein the transmembrane dimerization domain induces dimerization of the cell surface polypeptide, and the cell surface polypeptide binds to a predetermined antibody or a variant or fragment thereof. Also provided herein is a polynucleotide sequence encoding the polypeptide construct described herein.

[0011] Provided herein are methods and compositions that include a cell tag comprising a truncated variant of a polypeptide such as HER1, CD20, LNGFR, and CD52. In some cases, the truncated variant of the polypeptide does not bind to the endogenous receptor. The disclosed truncated non-immunogenic polypeptide can be used as a cell tag, for example, a cell marker, a starvation marker, or a killing tag.

[0012] As used herein, provided are compositions comprising engineered cells that express a polypeptide construct or polynucleotide described herein. Optionally, the engineered cells further express at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and / or a cytokine. As used herein, provided is a method of modulating the activity of engineered cells in a subject (e.g., receiving immunotherapy), the method comprising providing to the subject engineered cells encoding a polynucleotide construct disclosed herein, and further providing to the subject a predetermined binding partner that binds to the cells and modulates their activity. Also provided are systems and kits for use in the method. As used herein, provided is a polypeptide construct comprising a cell surface polypeptide and a transmembrane dimerization domain, wherein the transmembrane dimerization domain induces dimerization of the cell surface polypeptide, and the cell surface polypeptide binds to a predetermined antibody or a variant or fragment thereof. In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8

[0013] In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8

[0014] In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8

[0015] In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 In some embodiments, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. Optionally, the cell surface polypeptide comprises a HER1 polypeptide, and the HER1 polypeptide has at least 70%, 75%, 80%, 8 A polypeptide sequence having 5%, 90%, 95%, 99%, or 99.5% identity is included. In some cases, the HER1 polypeptide includes a polypeptide sequence including the sequence shown in SEQ ID NO: 211, 212, 213 , 214, 215, 216, or 217 .

[0016] In some embodiments, the cell surface polypeptide includes a CD20 polypeptide, and the CD20 polypeptide includes a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220 . Optionally, the CD20 polypeptide includes a polypeptide sequence including the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220 .

[0017] In some embodiments, the cell surface polypeptide includes an LNGFR polypeptide, and the LNGFR polypeptide includes a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 99.5% identity to the sequence shown in SEQ ID NO: 156, SEQ ID NO: 158, or SEQ ID NO: 16 0. Optionally, the cell surface polypeptide includes an LNGFR polypeptide, and the LNGFR polypeptide includes a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 9 9.5% identity to the sequence shown in SEQ ID NO: 156, SEQ ID NO: 158, or SEQ ID NO: 160 .

[0018] In some cases, the cell surface polypeptide does not bind to an endogenous receptor. Optionally​​ That is, the transmembrane dimerization domain can form a homodimer or a heterodimer together with a complementary dimerization domain. In some cases, the transmembrane dimerization domain contains at least one cysteine residue. In some embodiments, the transmembrane dimerization domain is a glycophorin A transmembrane domain or a fragment or variant thereof, a glycophorin A-integrin β3 chimeric transmembrane domain or a fragment or variant thereof, or a CD3 zeta transmembrane domain. In some cases, such a cell surface polypeptide contains at least a HER1 polypeptide. In some cases, the polypeptide construct is expressed in engineered cells. In some embodiments, the engineered cells are animal cells. In some cases, the animal cells are human cells. In some embodiments, the human cells are T cells or NK cells. In some cases, the engineered cells further contain a Sleeping Beauty transposase. In some cases, the engineered cells further express at least one additional exogenous polypeptide. In some cases, the engineered cells further express at least one exogenous receptor polypeptide or a fragment thereof. In some cases, the engineered cells further express at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some embodiments, the engineered cells further express at least one CAR, and the CAR is CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2,

[0019]

[0020] ​​​​​​​​​​​​​​​HER3, folate-binding protein, GD2, GD3, IL-13R-a2, KDR, ED B-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1 , h5T4, PSMA, TAG-72, EGFRvIII, CD123, and VEGF -R2 binds to at least one of them.

[0021] Optionally, the engineered cell further expresses at least one recombinant cytokine . In some cases, the recombinant cytokine includes at least one of IL-15, mbIL-15, IL- 2, IL-12, and IL-21. In some embodiments , the polypeptide construct is encoded by a polynucleotide integrated into the engineered cell by genome editing . Optionally, the genome editing includes the use of at least a site-specific serine recombinase system. In some cases, the polypeptide construct includes a linker that fuses the cell surface polypeptide with the transmembrane dimerization domain . Optionally, the homodimer or heterodimer of the polypeptide includes the polypeptide construct .

[0022] A polypeptide construct comprising a non-immunogenic cell surface polypeptide fused with a transmembrane dimerization domain, wherein the transmembrane dimerization domain induces dimerization of the cell surface polypeptide, and a polynucleotide encoding the polypeptide construct are provided. In some embodiments, the cell surface polypeptide does not bind to an endogenous receptor. Optionally , the cell surface polypeptide does not contain any endogenous signaling or trafficking function. Optionally, the cell surface polypeptide does not contain any endogenous signaling or trafficking function.

[0023] In some cases, the polynucleotide encodes at least one heterologous gene and comprises at least one sequence. In some embodiments, the at least one heterologous gene is modulated by an inducible promoter. Optionally, the at least one heterologous gene comprises at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine . In some embodiments, the at least one heterologous gene comprises the CAR, and the CAR binds to at least one of CD19, CD33, BCMA, CD44 , α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40 , HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, M AGE-A1, h5T4, PSMA, TAG-72, EGFRvIII, CD123, and VEGF-R2.

[0024] In some embodiments, the at least one heterologous gene comprises a cytokine. Optionally, the cytokine comprises at least one of IL-15, IL-2, IL-12, IL-21, and a fusion of IL-15 and IL-15Rα . In some embodiments, the cytokine is in a secreted form. In some cases, the cytokine is in a membrane-bound form.

[0025] In some embodiments, the polynucleotide comprises a polypeptide linker selected from the group consisting of 2A, GSG-2A, GSG linker (SEQ ID NO: 16), SGSG linker (SEQ ID NO: 18), furin linker mutants, and their derivatives, and comprises at least one comprises an array. In some embodiments, the 2A linker is a p2A linker, a T2A linker, an F2A linker, or an E2A linker.

[0026] In some cases, the polypeptide construct acts as a tag that either enriches cells, selects cells, or induces cell death in cells expressing the cell surface molecule. Optionally, the cell surface polypeptide comprises at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. In some embodiments, the transmembrane dimerization domain comprises a glycophorin A transmembrane domain or a fragment or variant thereof, a glycophorin A-integrin β3 chimeric transmembrane domain or a fragment or variant thereof, or a CD3 zeta transmembrane domain.

[0027] Optionally, the vector comprises a polynucleotide. In some cases, the vector is a lentiviral vector, a retroviral vector, or a non-viral vector. In some embodiments, the non-viral vector is a Sleeping Beauty transposon. Optionally, the polynucleotide is integrated into the engineered cell by genome editing. Optionally, the genome editing includes the use of at least a site-specific serine recombinase system.

[0028] A method of modulating the activity of genetically engineered cells in a subject, the method comprising providing to the subject an amount of genetically engineered cells encoding a polypeptide construct comprising a cell surface polypeptide fused to a transmembrane dimerization domain, wherein the transmembrane dimerization domain inducing dimerization of the cell surface polypeptide, wherein the cell surface polypeptide binds to a predetermined binding partner or a variant or fragment thereof; binding to the genetically engineered cell, thereby providing the predetermined binding partner to the subject in an amount sufficient to modulate the activity of the genetically engineered cell. A method is provided that includes the steps of.

[0029] In some embodiments, the cell surface polypeptide is a non-immunogenic polypeptide. In some embodiments, the cell surface polypeptide includes at least one of a HER1 polypeptide, an LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. In some cases, the cell surface polypeptide does not bind to an endogenous receptor . Optionally, the transmembrane dimerization domain can form a homo-dimer or a hetero-dimer with a complementary dimerization domain. In some embodiments, the transmembrane dimerization domain includes at least one cysteine residue. In some cases, the transmembrane dimer ization domain is a glycophorin A transmembrane domain or a fragment or variant thereof, a glycophorin A-integrin β3 chimeric transmembrane domain or a fragment or variant thereof, or alternatively includes a CD3 zeta transmembrane domain.

[0030] Optionally, the binding partner includes an antibody or a cell surface polypeptide-binding region thereof. In some embodiments, the antibody includes at least one of a monoclonal antibody, an scFv, an scF ab, a diabody, and a camelid antibody. In some cases, the antibody is rituximab, cetuximab, alemtuzumab, pa nimuzumab, or trastuzumab. and necitumumab.

[0031] In some cases, the genetically engineered cells are at least one of T cells and NK cells. In some embodiments, at least one of the genetically engineered cells comprises at least In some cases, the gene further expresses another exogenous polypeptide. At least one of the engineered cells is a chimeric antigen receptor (CAR), a T cell receptor (TC R), and a cytokine. At least one of the engineered cells further expresses at least one CAR; The CAR can be any of the following: CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate Synthetic protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelial , CD22, EGFR, MUC-1, MUC-16, MAGE-A1, h5T4, PS MA, TAG-72, EGFRvIII, CD123, and VEGF-R2 At least one of them will be combined.

[0032] In some embodiments, at least one of the genetically engineered cells comprises at least one In some instances, the recombinant cytokine further expresses At least one of IL-15, mbIL-15, IL-2, IL-12, and IL-21 Optionally, the predetermined binding partner is a cytokine storm or or a systemic inflammatory response. provided in an amount. In some embodiments, the predetermined binding partner is provided in an amount sufficient to cause a reduction of at least one symptom associated with tumor lysis syndrome. In some cases the polypeptide construct is encoded by a polynucleotide integrated into the engineered cell by genome editing. In some cases the genome editing involves the use of at least a site specific serine recombinase system.

[0033] A polynucleotide encoding a truncated non-immunogenic CD20 polypeptide that binds to an anti-CD20 antibody, wherein the truncated non-immunogenic CD20 polypeptide does not bind to an endogenous receptor, is provided.

[0034] In some embodiments, the CD20 polypeptide comprises a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the sequence shown in SEQ ID NO: 109. In some cases, the CD20 polypeptide comprises a polypeptide sequence comprising the sequence of SEQ ID NO: 109. In some cases, the CD20 polypeptide binds to the anti-CD20 antibody with a binding efficiency of at least 50%, 60%, 7 0%, 80%, or 90% of native CD20. In some embodiments the anti-CD20 antibody comprises at least one of rituximab, cetuximab, tositumomab, belimumab, afutuzumab, brontezumab, and obinutuzumab.

[0035] At least the HER1 domain III or a fragment thereof and a truncated HER1 domain ​​​​​​A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells.

[0036] In some embodiments, the truncated HER1 domain IV comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40% A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. %, 45%, or 50% truncation of the HER1 domain IV.

[0037] Optionally, the truncated HER1 domain IV has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the polypeptide sequence shown in SEQ ID NOs: 203, 204, 205, 206, 207, 208, or 209. In some cases, the truncated HER1 domain IV comprises a polypeptide sequence comprising the sequence shown in SEQ ID NOs: 203, 204, 205, 206, 207, 208, or 209. In some embodiments, the HER1 domain III or a fragment thereof has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the polypeptide sequence shown in SEQ ID NO: 200. Optionally, the HER1 domain III comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 200. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells.

[0038] In some cases, the polynucleotide comprises a CD28 transmembrane domain and a peptide linker for conjugating the HER1 polypeptide to the CD28 transmembrane domain. A polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of IV, wherein the HER1 polypeptide binds to an anti-HER1 antibody and the HER1 polypeptide is expressed in engineered cells. Further code. Optionally, the polynucleotide encodes a polypeptide construct comprising a polypeptide sequence comprising the sequence shown in SEQ ID NO: 57. In some embodiments the anti-HER1 antibody comprises at least one of rituximab, cetuximab, furtuximab, depatuxizumab umab, imigatuzumab, laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab umab, and zalutumumab. A polynucleotide encoding a truncated non-immunogenic CD52 polypeptide that binds to an anti-CD52 antibody, wherein the truncated non-immunogenic CD52 polypeptide does not bind to an endogenous receptor and the CD52 polypeptide is expressed in a manipulated cell, is provided.

[0039] Optionally, the CD52 polypeptide comprises a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99. 5% identity to the sequence shown in SEQ ID NO: 143. In some cases, the CD52 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 143. In some embodiments, the polynucleotide is expressed in a cell further comprising at least one sequence encoding at least one heterologous gene. In some embodiments, the at least one heterologous gene is modulated by an inducible promoter. In some cases, the at least one heterologous gene comprises at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some embodiments

[0040] 5% identity to the sequence shown in SEQ ID NO: 143. In some cases, the CD52 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 143. 5% identity to the sequence shown in SEQ ID NO: 143. In some cases, the CD52 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 143.

[0041] In some embodiments, the polynucleotide is expressed in a cell further comprising at least one sequence encoding at least one heterologous gene. In some embodiments, the at least one heterologous gene is modulated by an inducible promoter. In some cases, the at least one heterologous gene comprises at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some embodiments one heterologous gene is modulated by an inducible promoter. In some cases, the at least one heterologous gene comprises at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some embodiments one heterologous gene is modulated by an inducible promoter. In some cases, the at least one heterologous gene comprises at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some embodiments the at least one heterologous gene comprises at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some embodiments ​​, the at least one heterologous gene includes a CAR, and the CAR binds to at least one of CD19, CD3 3, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, E GP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3 , IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC -1, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRv III, CD123, and VEGF-R2.

[0042] In some embodiments, the at least one heterologous gene includes a cytokine. In some embodiments, the cytokine includes at least one of IL-15, IL-2, IL-12, IL-21 , and a fusion of IL-15 and IL-IL-15Rα. Optionally, the cytokine is in a secreted form. In some cases, the cytokine is in a membrane-bound form.

[0043] In some embodiments, the vector includes the polynucleotide. Optionally, the vector is a lentiviral vector, a retroviral vector, or a non-viral vector . In some cases, the non-viral vector is a Sleeping Beau ty transposon. In some embodiments, the polynucleotide is integrated into the engineered cell by genome editing . In some cases, the genome editing includes the use of at least a partial site-specific serine recombinase system. Optionally, the engineered cell encodes the polynucleotide . Optionally, the engineered cell is a T cell or an NK cell. Optionally, the polynucleotide encodes a polypeptide .

[0044] Further provided herein is a method of treating cancer, comprising administering to a subject an effective amount of a polynucleotide. In some embodiments, a method is provided that includes administering engineered cells that contain an otide. The method further comprises the step of: detecting at least one polypeptide capable of binding to a polypeptide expressed on the engineered cell; Optionally, the method further comprises administering a binding partner of is an antibody.

[0045] Provided herein is a method of modulating the activity of a genetically engineered cell in a subject, the method comprising: a first truncated non-immunogenic polypeptide and a second truncated non-immunogenic polypeptide. A polypeptide comprising a cell surface polypeptide which is a chimeric polypeptide comprising a functional polypeptide and a providing a quantity of genetically engineered cells encoding a tid construct, said cells The cell surface polypeptide is capable of binding to at least one predetermined binding partner or a variant or variant thereof. and binding to said fragments to said engineered cells, thereby inhibiting their activity. providing said predetermined binding partner to said subject in an amount sufficient to regulate the sex of said subject. A method is presented that includes:

[0046] In some embodiments, the first truncated non-immunogenic polypeptide is Optionally, said second truncation comprises a fragment or derivative of a member of said family. The non-immunogenic polypeptides include fragments or derivatives of EGFR family members. In some instances, the first truncated non-immunogenic polypeptide is a HER1 polypeptide. In some embodiments, the HER1 polypeptide comprises the peptide sequence of SEQ ID NO: 211. 、at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the sequences shown in 212, 213, 214, 215, 216, or 217. In some embodiments, the HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 211, 212, 213, 214, 215, 216, or 217. Optionally, the HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 211. at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity. In some embodiments, the HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 211, 212, 213, 214, 215, 216, or 217. Optionally, the HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 211. shown in SEQ ID NO: 211, 212, 213, 214, 215, 216, or 217. Optionally, the HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 211. Optionally, the HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 211. shown in SEQ ID NO: 211.

[0047] Optionally, the second truncated non-immunogenic polypeptide comprises at least one of a HER2 polypeptide, an ErbB3 polypeptide, and an ErbB4 polypeptide. In some embodiments, the polypeptide construct comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the sequences shown in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, or SEQ ID NO: 105. shown in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, or SEQ ID NO: 105. shown in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, or SEQ ID NO: 105. shown in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, or SEQ ID NO: 105. at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity.

[0048] In some embodiments, the at least one predetermined binding partner binds to the HER1 polypeptide. In some cases, the at least one predetermined binding partner comprises at least one of rituximab, cetuximab, panitumumab, trastuzumab, pertuzumab, ramucirumab, nimotuzumab, matuzumab, necitumumab, and zalutumumab. Optionally, the at least one predetermined binding partner binds to the second truncated non-immunogenic polypeptide. In some cases, the at least one predetermined binding partner comprises at least one of rituximab, cetuximab, panitumumab, trastuzumab, pertuzumab, ramucirumab, nimotuzumab, matuzumab, necitumumab, and zalutumumab. Optionally, the at least one predetermined binding partner binds to the second truncated non-immunogenic polypeptide. rituximab, cetuximab, panitumumab, trastuzumab, pertuzumab, ramucirumab, nimotuzumab, matuzumab, necitumumab, and zalutumumab. rituximab, cetuximab, panitumumab, trastuzumab, pertuzumab, ramucirumab, nimotuzumab, matuzumab, necitumumab, and zalutumumab. Optionally, the at least one predetermined binding partner binds to the second truncated non-immunogenic polypeptide. Optionally, the at least one predetermined binding partner binds to the second truncated non-immunogenic polypeptide. It further includes a predetermined binding partner. In some cases, the second truncated non-immunogenic polypeptide is a HER2 polypeptide, and the second predetermined binding partner is pertuzumab.

[0049] In some cases, the polypeptide construct further includes a signal peptide. In some cases, the polypeptide construct further includes a transmembrane domain. In some embodiments the transmembrane domain includes a transmembrane dimerization domain. In some cases, the transmembrane dimerization domain includes a glycophorin A transmembrane domain, a glycophorin A-integrin β 3 chimeric transmembrane domain, or a CD3 zeta transmembrane domain. In some cases, the aforementioned transmembrane dimerization domain has at least 70%, 75%, 80%, 85%, 90% identity with the polypeptide sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32, 95%, 99%, or 99.5%. In some embodiments, the transmembrane dimerization domain includes a polypeptide sequence including the sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32.

[0050] In some cases, the first truncated non-immunogenic polypeptide includes a CD20 poly peptide. In some embodiments, the CD20 polypeptide has at least 70%, 7 5%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220, and includes a po lypeptide sequence. In some cases, the CD20 polypeptide is SEQ ID NO: 218, It includes a polypeptide sequence containing the sequence shown in SEQ ID NO: 219 or SEQ ID NO: 220. In some embodiments, the at least one predetermined binding partner is rituximab, tr situmumab, pertuzumab, afucosylated anti-HER2 antibody, brontezumab, and obinutuzumab including at least one of them.

[0051] A truncated non-immunogenic HER1 polypeptide consisting of HER1 domain III and truncated HER1 domain IV, which binds to an anti-HER1 antibody polypeptide; a CD28 transmembrane domain; and a peptide linker for conjugating the HER1 polypeptide to the CD28 transmembrane domain, and a polypeptide construct is provided that is expressed in engineered cells.

[0052] In some embodiments, the HER1 domain III includes a polypeptide sequence containing the sequence shown in SEQ ID NO: 200 and the HER1 domain IV includes a polypeptide sequence containing the sequence shown in SEQ ID NO: 203. In some cases, the CD28 transmembrane domain includes a polypeptide sequence containing the sequence shown in SEQ ID NO: 36, and the peptide linker includes a G4S peptide linker (SEQ ID NO: 221). Optionally, the G4S peptide linker (SEQ ID NO: 221) includes a polypeptide sequence containing the sequence shown in SEQ ID NO: 22. In some embodiments, the polypeptide construct includes a polypeptide sequence containing the sequence shown in SEQ ID NO: 57.

[0053] Optionally, the anti-HER1 antibody is rituximab, cetuximab, panitumumab, Depatuximab, Imgatuzumab, Laprituximab, Matuzumab, Necitumumab, Nitto Contains at least one of motuzumab, panitumumab, and zalutumumab.

[0054] Polypeptides containing truncated non-immunogenic CD20 polypeptides that bind to anti-CD20 antibodies A peptide construct is provided, the polypeptide construct being expressed in an engineered cell.

[0055] In some embodiments, the polypeptide construct has a sequence similar to that shown in SEQ ID NO:109. at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 9.5% identity. Optionally, the polypeptide construct has SEQ ID NO: 109. In some instances, the anti-CD20 antibody comprises a polypeptide sequence comprising the sequence shown in , rituximab, tositumomab, veltuzumab, afutuzumab, brontuzumab, and and obinutuzumab.

[0056] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the present invention and the advantages thereof will be readily apparent from the following detailed description of exemplary embodiments in which the principles of the present disclosure are employed. The foregoing can be obtained by reference to the following detailed description and accompanying drawings, which set forth: [Brief description of the drawings]

[0057]

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Mode for Carrying Out the Invention

[0058] The following description and examples illustrate in detail embodiments of the present invention. The present invention is not limited to the specific embodiments described in this specification and is intended to be variable as such. It should be understood that those skilled in the art will make numerous variations and modifications to the present invention, and these will be recognized as being included within its scope.

[0059] All terms are intended to be understood as they are understood by those skilled in the art. Unless otherwise specified, the technical terms and scientific terms used in this specification have the same meaning as those generally understood by those skilled in the technical field to which this disclosure pertains.

[0060] The subsection headings used in this specification are for structural purposes only and the main It should not be considered as limiting the subject matter.

[0061] The various features of the present invention may be described in the context of a single embodiment, but the features may also be presented individually or in any suitable combination. Conversely, although the present invention may be described herein for clarity in the context of individual embodiments, the present invention can also be practiced in a single embodiment. The following definitions supplement those in the art and are directed to this application and do not belong to any related or unrelated cases, such as any jointly owned patents or applications. Any methods and materials similar or equivalent to those described herein may be used in the practice of the disclosed tests, but the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In this application, unless otherwise stated, the use of the singular includes the plural. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. In this application, unless stated otherwise, the use of "or" means "and / or." Further, the use of terms such as "including" and other forms such as "include," "includes," and "included" is not limiting.

[0062]

[0063] ​

[0064] As used herein, references to "some embodiments", "an embodiment", "one embodiment", or "other embodiments" mean that the particular features, structures, or characteristics described in connection with the embodiments are included in at least some embodiments of the invention, but not necessarily in all embodiments.

[0065] As used in this specification and the claims, the terms "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "include" and "includes"), or "containing" (and any form of "containing", such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any embodiment discussed herein can be practiced with respect to any method or composition of the invention, and vice versa is assumed to be true. Further, the methods of the invention can be achieved using the compositions of the invention. The term "about" and its grammatical equivalents, as used herein with respect to a reference numerical value and its grammatical equivalents, can include the numerical value itself and values within the range of the numerical value ±10%. For example,

[0066] ​ If so, the amount of "about 10" includes any amount from 9 to 11. For example, in relation to the reference value the term "about" also includes a range of the value ± 10%, 9%, 8%, 7%, 6%, 5% from this value, 4%, 3%, 2%, or 1%.

[0067] "Isolated" means the extraction of nucleic acid from its natural environment. "Purified" means that a given nucleic acid is either of natural origin (including genomic DNA and mRNA), synthetic (including cDNA), and / or amplified under laboratory conditions, and the purity is increased, where "purity" is a relative term and not "absolute purity" in this sense. On the other hand, nucleic acids and proteins may be formulated with diluents or adjuvants, but for practical purposes, it should be understood that they can be considered isolated. For example, nucleic acids are typically mixed with acceptable carriers or diluents when used for introduction into cells.

[0068] As used herein, "polynucleotide" or "oligonucleotide" refers to a polymer form of nucleotides of any length that are ribo nucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded DNA and single-stranded DNA, triple-stranded DNA, as well as double-stranded RNA and single-stranded RNA in addition to. This term also includes, for example, modified forms of polynucleotides by methylation and / or capping as well as unmodified forms of polynucleotides. The term also intends to include molecules containing nucleotide analogs in addition to nucleotides that are not of natural origin or synthetic nucleotides. ​​

[0069] The term "polypeptide" is used interchangeably with the terms "polypeptide" and "protein" and refers to a polymer of amino acid residues. A "mature protein" is a full-length protein that optionally includes glycosylation or other modifications typical of a protein in a given intracellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can include one or more synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxy-phenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine

[0070] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can include one or more synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxy-phenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine It contains phenylalanine and α-tert-butylglycine. This disclosure relates to the expression of the polypeptides described herein within engineered cells and further contemplates that the expression of the polypeptides described herein may be associated with post-translational modification of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include acylations such as lipidation, acetylation, and formylation, glycosylations (including N-linked and O-linked), amidation, hydroxylation, alkylations such as methylation and ethylylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0071] As used herein, "antibody" refers to a monoclonal or polyclonal antibody. A whole antibody typically consists of four polypeptides: two identical copies of the heavy (H) chain polypeptide and two identical copies of the light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and C H3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH region and the VL region have a common general structure that includes four framework regions where the sequences of these regions are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable regions" of the antibody that contribute to binding to the antigen.

[0072] ​​​​ "Antigen recognition portion or domain" refers to a molecule or a portion of a molecule that specifically binds to an antigen. In some embodiments, the antigen recognition portion is an antibody, an antibody-like molecule, or a fragment thereof and the antigen is a tumor antigen.

[0073] "Antibody-like molecule" can be, for example, a protein that is a member of the Ig super -family and can selectively bind to a partner. MHC molecules and T cell receptors are such molecules. In some embodiments, the antibody-like molecule is a TCR. In some em bodiments, the TCR is modified to increase its binding affinity to its MHC .

[0074] As used herein, the terms "fragment of an antibody", "antibody fragment", "functional fragment of an antibody", and "antigen -binding portion" are used interchangeably to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., N at. Biotech., 23(9):1126-1129 (2005)). Antibody fragments can include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof as desired. Examples of antibody fragments are: (i) Fab fragments, which are monovalent fragments consisting of the VL domain, VH domain, CL domain, and CH1 domain; ( ii) F(ab’)2 fragments, which are divalent fragments containing two Fab fragments linked by a disulfide bridge in the stalk region; (iii) Fv fragments, which consist of the VL domain and VH domain of a single arm of an antibody; (iv) single polypeptide s consisting of two domains ; ( v) single polypeptide s consisting of two domains The two domains of the Fv fragment are joined by a synthetic linker that allows them to be synthesized as a single chain. Single-chain Fv (scFv) ( For example, Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Ac. ad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 77 8 (1998); and (v) each polypeptide chain is to the VL via a peptide linker that is too short to allow pairing between the VH and VL of A dimeric molecule comprising linked VHs and thus having two functional antigen-binding sites. The complementary domains on the different VH-VL polypeptide chains are paired to generate a complementary domain. diabodies, which are dimers of polypeptide chains that move in a circular fashion. Antibody fragments are known in the art and are described, for example, in U.S. Pat. No. 8,603,950. This is described in more detail in the specification.

[0075] Nucleic acids and / or nucleic acid sequences may be derived from natural or artificial common ancestral nucleic acids or ancestral nucleic acid sequences. A protein and / or protein sequence is "homologous" if it is derived from the sequence. their coding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or ancestral nucleic acid sequence Homologous molecules are sometimes referred to as homologs. For example, Any naturally occurring protein may be modified by any available mutagenesis method. When expressed, this mutant nucleic acid produces the protein encoded by the original nucleic acid. encodes a polypeptide that is homologous thereto. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or their sequences). The exact percentage of identity between sequences useful for establishing homology varies with the nucleic acids and proteins in question, but homology is established using at least 25% sequence identity. High levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95 %, or 99%, or more sequence identity can also be used to establish homology. In the context of two nucleic acid sequences or amino acid sequences of a polypeptide, the term "identical" or "sequence identity" refers to residues within two sequences that are the same when aligned for maximum correspondence

[0076] over a specified comparison region. In some embodiments, a polypeptide herein is, for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or a fragment thereof, as measured by, e.g., BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described in reference to a starting nucleic acid, e.g., they can be, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference nucleic acid or a fragment thereof, as measured by, e.g., BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. One molecule is, with respect to a large molecule, a particular ... ... For example, they can be, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 9 9%, or 100% identical to a reference nucleic acid or a fragment thereof, as measured by, e.g., BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. It is possible that one molecule is, with respect to a large molecule, a particular When we say that there is a percentage of sequence identity, this means that the two molecules are optimally aligned. If so, then that percentage of residues in the small molecule are in the order in which the two molecules are optimally aligned. This means finding matching residues within a larger molecule, according to the sequence.

[0077] A "transposon" or "transposable element" (TE) is a gene that determines its location in the genome. Change, possibly creating or storing mutations, and altering the genome size of the cell , the vector DNA sequence. Transposition often results in duplication of the TE. Class I TEs are copied in two steps: first, class I TEs are copied from DNA to RNA. The resulting RNA is then reverse transcribed into DNA. The replicated DNA is inserted into a new location in the genome. The transcription factor is catalyzed by the reverse transcriptase enzyme, which can be encoded by the genome. , as well as retroviruses such as HIV. The transposition mechanism does not involve an RNA intermediate. Transposition is mediated by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA. whereas other transposases bind to specific DNA sequence targets. The enzyme produces staggered cleavages at the target site, resulting in 5 This results in single-stranded DNA overhangs (sticky ends) at the ' or 3' ends. The first step is to excise the DNA transposon, which then transforms it into a new The new target site is then ligated to the target site, a process that involves DNA polymerisation to fill the gap. It involves the enzyme activity and the DNA ligase activity that joins the sugar-phosphate backbone. The insertion site of a DNA transposon is located at the target DNA site, resulting in duplication of the target site. A can be created by staggered cuts in A and filling in the gaps with DNA polymerase. short direct repeats followed by excision of the TE by transposase During the S phase of the cell cycle, the donor site is identified by a series of inverted repeats that are important for Transposition occurs when the target site has not yet been replicated, but the target site has already been replicated. The translocation and paste TE are overlapping. The metastasis is classified into class I TE and class II TE. In either case, they can be classified as either "autonomous" or "non-autonomous". While E can move on its own, non-autonomous TEs require the presence of another TE to move. This is because non-autonomous TEs require a transposase (in the case of class II) or This is often due to a lack of reverse transcriptase (in the case of class I).

[0078] "Transposase" refers to a gene that binds to the ends of transposons and functions as a cut-and-paste enzyme. Catalyzes the movement of a transposon to another part of the genome by a replicative or replicative transfer mechanism. In some embodiments, the catalytic activity of a transposase is used to transfect genes into It can be moved from a vector into the genome.

[0079] The nucleic acid sequences and vectors disclosed or contemplated herein can be "transfected" into cells. Introduced by "transfection," "transformation," "nucleofection," or "transduction" As used herein, "transfection," "transformation," or Or "transfection" refers to the introduction of one or more exogenous polynucleotides into a host cell by using a physical method or a chemical method. In the art, many transfection methods are known. For example, see calcium phosphate DNA coprecipitation method (e.g., refer to Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expr ession Protocols, Humana Press (1991)); DEAE-dextran method ; electroporation method; cationic liposome-mediated transfection method; tungsten particle -facilitated gene gun method (Johnston, Nature, 346: 776-777 (1990)); strontium phosphate co -precipitation method (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)); and nucleofection (Trompeter et al., J. Immunol. Methods 274:245-256 (2003)). Phage vectors or viral vectors can be introduced into host cells after growing infectious particles in appropriate packaging cells, many of which are commercially available. As used herein, "tumor antigen" refers to any antigenic substance produced or overexpressed in tumor cells. Tumor antigens can, for example, induce an immune response in a host. Alternatively, for the purposes of the present disclosure, a tumor antigen can be a protein that is expressed in both normal cells and tumor cells but is an appropriate therapeutic target for identifying a particular tumor type.

[0080]

[0081] "Promoter" refers to a region of a polynucleotide that induces transcription of a coding sequence. The promoter is in the vicinity of the transcription start site of a gene on the same strand of DNA and is located upstream (towards the 5'-side region of the sense strand). Since a certain promoter is active in all situations within the cell, it is constitutive, whereas other promoters, for example, inducible promoters are regulated and become active in response to specific stimuli.

[0082] The term "promoter activity" refers to the degree of expression of a nucleotide sequence operably linked to the promoter whose activity is being measured. Promoter activity can be measured directly, for example, by determining the amount of RNA transcript produced by Northern blot analysis, and can also be measured indirectly by determining the amount of product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0083] As used herein, "inducible promoter" refers to a promoter that is induced to activity by the presence or absence of a transcriptional regulator, for example, a biotic factor or an abiotic factor. Inducible promoters are useful because the expression of genes operably linked to them can be switched on or off at a particular stage of the development of an organism or, in particular, a tissue. Examples of inducible promoters are alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature- regulated promoters, and light-regulated promoters. In some embodiments, the inducible promoter is part of a gene switch.

[0084] As used herein, the term "enhancer" refers to, for example, a DNA sequence that increases the transcription of an operably linked nucleic acid sequence. An enhancer can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. In the art, numerous enhancers from a variety of different sources are well known and are available as cloned polynucleotides or within cloned polynucleotides (e.g., from other commercial or personal sources in addition to depositories such as the ATCC). Many polynucleotides, including promoters (such as the commonly used CMV promoter), also contain enhancer sequences. Enhancers can be located upstream of the coding sequence, within it, or downstream of it. The term "Ig enhancer" refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (mu) 5' enhancer, the light chain (kappa) 5' enhancer, the kappa intron enhancer and the mu intron enhancer, as well as the 3' enhancer) (see generally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, Raven Press, New Y ork (1993), pages 353-363, and U.S. Patent No. 5,885,827). The term "coding sequence" as used herein refers to a polynucleotide that encodes a polypeptide and can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. In the art, numerous enhancers from a variety of different sources are well known and are available as cloned polynucleotides or within cloned polynucleotides (e.g., from other commercial or personal sources in addition to depositories such as the ATCC). Many polynucleotides, including promoters (such as the commonly used CMV promoter), also contain enhancer sequences. Enhancers can be located upstream of the coding sequence, within it, or downstream of it. The term "Ig enhancer" refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (mu) 5' enhancer, the light chain (kappa) 5' enhancer, the kappa intron enhancer and the mu intron enhancer, as well as the 3' enhancer) (see generally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363, and U.S. Patent No. 5,885,827).

[0085] As used herein, the term "coding sequence" refers to a polynucleotide that encodes a polypeptide Refers to a segment of DNA. In the vicinity of the 5' end, the start codon binds to it, and in the vicinity of the 3' end, the stop codon binds. The coding sequence can also be referred to as an open reading frame.

[0086] As used herein, "operably linked" refers to a physical and / or functional linkage of a DNA segment to another DNA segment such that the segments function in their intended manner. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence in a manner that allows modulation of the transcription of the DNA sequence, either directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream of the transcription start site of the promoter, within the proper reading frame relative to the transcription start site, such that elongation of transcription can proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when it is ligated to the DNA sequence such that it increases or decreases the transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of the DNA sequence. When a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the signal sequence, for example, a promoter, enhancer, and / or silencer, etc., in a manner that allows direct or indirect modulation of the transcription of the DNA sequence. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream of the transcription start site of the promoter, within the proper reading frame relative to the transcription start site, such that elongation of transcription can proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when it is ligated to the DNA sequence such that it increases or decreases the transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of the DNA sequence. When a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the signal sequence is operably linked to the DNA sequence encoding the polypeptide such that the polypeptide is secreted. sequence is ligated to the DNA sequence encoding the polypeptide in a way that enables the polypeptide to be secreted. When the DNA sequence is ligated to the promoter at the transcription start site of the promoter downstream, within the proper reading frame relative to the transcription start site, and allows elongation of transcription to proceed through the DNA sequence, it is operably linked to the promoter. An enhancer or silencer is operably linked to the DNA sequence encoding the gene product when it is ligated to the DNA sequence so that it can increase or decrease the transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of the DNA sequence. When a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the signal sequence is operably linked to the DNA sequence encoding the polypeptide such that the polypeptide is secreted. sequence is ligated to the DNA sequence encoding the polypeptide so that the polypeptide can be secreted. When an enhancer or silencer is ligated to the DNA sequence encoding the gene product so that it can increase or decrease the transcription of the DNA sequence, respectively, it is operably linked to the DNA sequence encoding the gene product. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of the DNA sequence. When a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the signal sequence is operably linked to the DNA sequence encoding the polypeptide such that the polypeptide is secreted. sequence may be located upstream, downstream, or embedded within the coding region of the DNA sequence. When a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the signal sequence is operably linked to the DNA sequence encoding the polypeptide such that the polypeptide is secreted. The DNA of the r array is operably linked to the DNA encoding the polypeptide. DN Ligation of the A array to the regulatory sequence is typically by ligation at appropriate restriction sites or is achieved via an adapter or linker inserted within the sequence using restriction endonucleases known to those skilled in the art.

[0087] The term "transcription regulator" refers to a biochemical element (e.g., a repressor protein or a nuclear inhibitory protein) that acts to prevent or inhibit the transcription of a promoter-driven DNA sequence under certain environmental conditions, or a biochemical element (e.g., an inducer or enhancer) that acts to allow or stimulate the transcription of a promoter-driven DNA sequence under certain environmental conditions.

[0088] The term "induction" refers to an increase in the transcription of a nucleic acid sequence, promoter activity, and / or promoter expression brought about by a transcription regulator compared to some transcriptional basal level.

[0089] A "target" gene or "heterologous" gene or "gene of interest (GOI)" refers to a gene introduced into a host cell by gene transfer.

[0090] As used herein, "recombinase" refers to a group of enzymes that can facilitate site-specific recombination between defined sites, where the sites are physically separated on a single DNA molecule or where the sites are present on separate DNA molecules. The DNA sequences of the defined recombination sites are not necessarily identical. The initiation of recombination is by protein-DNA interaction ​​​​​​​​​Depending on the interaction, within the enzyme group, there are phage integration and excision (e.g., λ integrase, ΦC31), resolution of circular plasmids (e.g., Tn3, γδ, Cre, Flp), inversion of DNA for alternative gene expression (e.g., Hin, Gin, Pin), assembly of genes (e.g., nitrogen fixation genes by Anabaena) during development, and transfer (e.g., IS607 transposon) catalyzed by a number of proteins. Most site-specific recombinases fall into one of two families based on evolutionary relatedness and mechanistic relatedness. These are either the λ integrase family or the tyrosine recombinases (e.g., Cre, Flp, XerD), and the resolvase / integrase family, or the serine recombinase family (e.g., ΦC31, TP901-1, Tn3, γδ). .

[0091] A "recombinant junction site" is a specific polynucleotide sequence recognized by the recombinase enzymes described herein. Typically, one site is present within a target nucleic acid (e.g., a eukaryotic or prokaryotic chromosome or episome), and another site is present on the nucleic acid to be integrated at the target recombination site. Two different sites (referred to as "complementary sites") are involved. Herein, the terms "attB" and "attP" are used to refer to the junction (or recombination) sites derived from the bacterial target and the phage donor, respectively, but the recombination sites for a particular enzyme may have different names. Recombination sites typically comprise a left arm and a right arm separated by a core region or a spacer region. ) are used, but the recombination sites for a particular enzyme may have different names. Recombination sites typically comprise a left arm and a right arm separated by a core region or a spacer region. Therefore, the attB recombination site consists of BOB’ [in the sequence, B and B’ are the left and right arms respectively, and O is the core region]. Similarly, attP is POP’ [in the sequence, P and P’ are arms, and O is also the core region here]. When recombining the attB site and the attP site, and during the simultaneous integration of nucleic acids at the target, the recombination sites sandwiching the DNA to be integrated are referred to as “attL” and “attR”. Therefore, using the above terminology, the attL site and the attR site consist of BOP’ and POB’’, respectively. In some of the representations in this specification, “O” is omitted, and attB and attP are referred to as BB’ and PP’, respectively. , the left and right arms, and O is the core region]. Similarly, at tP is POP’ [in the sequence, P and P’ are arms, and O is also the co re region here]. When recombining the attB site and the attP site, and during the simultaneous integration of nucleic acids at the target site, the recombination sites sandwiching the DNA to be integrated are referred to as “attL” and “attR”. Therefore, using the above terminology, the attL site and the attR site consist of BOP’ and POB’’, respectively. In some of the representations in this specification, “O” is omitted, and attB and attP are referred to as BB’ and PP’, respectively.

[0092] The term “gene editing” or “genome editing” refers to the insertion, deletion, or replacement of DNA nucleotides within the genome of an organism. Typically, genome editing uses engineered nucleases that can create site-specific double-strand breaks at predetermined positions in the genome. This disclosure contemplates any means for editing the genome. Non-limiting examples of genome editing methods include CRISPR, Argonaute, and the AttSite site-specific serine recombinase system. As used herein, the “CRISPR gene editing system” or “CRISPR system” refers to any RNA-guided Cas protein-mediated process for targeting changes in DNA sequences to specific regions of the genome. As used herein, the “Argonaute gene editing system” refers to a DNA sequence This disclosure contemplates any means for editing the genome. Non-limiting examples of genome editing methods include CRISPR, Argonaute, and the AttSite site-specific serine recombinase system. As used herein, the “CRISPR gene editing system” (CRISPR gene editing sy stem) or “CRISPR system” refers to any RNA-guided Cas protein-mediated process for targeting changes in DNA sequences to specific regions of the genome. As used herein, the “Argonaute gene editing system” refers to a DNA sequence to a specific region of the genome. As used herein, the “Argonaute gene editing system” refers to a DNA sequence mediated process for targeting changes in DNA sequences to specific regions of the genome. As used herein, the “Argonaute gene editing system” refers to a DNA sequence The change is directed to a specific region of the genome for targeting by any single-stranded DNA-induced type of Argonaute endonuclease-mediated process. As used herein, "Att Site gene editing system" or "site-specific serine recombinase gene editing system" or "site-specific serine recombinase system" refers to the steps of preparing a eukaryotic cell containing a first recombination junction site and a second recombination junction site; and contacting the first recombination junction site and the second recombination junction site with a prokaryotic cell recombinase polypeptide, resulting in recombination between the recombination sites, wherein the recombinase polypeptide is capable of mediating recombination between the first recombination junction site and the second recombination junction site, and the first recombination junction site is a phage genome recombination junction site (attP) or a bacterial genome recombination junction site (attB), and the second recombination junction site is attB or attP and, when the first recombination junction site is attB, the second recombination junction site is att P, and when the first recombination junction site is attP, the second recombination junction site is a ttB, and under the condition that the recombinase is selected from the group consisting of phage recombinases of Listeria monocytogenes, phage recombinases of Streptococcus pyogenes, phage recombinases of Bacillus subtilis, phage recombinases of Mycobacterium tuberculosis, and phage recombinases of Mycobacterium smegmatis, any process is meant. Examples of embodiments of the AttSite serine recombinase system are provided. is incorporated herein by reference in its entirety, in U.S. Patent No. 9,034,650 which is presented therein.

[0093] As used herein, the term " endogenous" when referring to a molecule such as a polynucleotide or polypeptide refers to the native form of the molecule that can be found in a wild-type cell or organism. A molecule that is found endogenously in an organism can typically be contrasted with an engineered molecule, which is not native and is described herein. For example, an engineered molecule can include a variant of a native polypeptide or polynucleotide. In some embodiments, a variant of a native polypeptide is a truncated variant of the native polypeptide. As used herein, the term "truncated variant" refers to a protein or polypeptide in which one or more sequences and / or domains of amino acids are missing as compared to the endogenous form of the protein or polypeptide. For example, a truncated variant incorporated into a polypeptide construct described herein may often be missing the sequence of amino acids corresponding to a domain (e.g., an intracellular signaling domain, a transmembrane domain, a ligand-binding domain, etc.) that is present in the endogenous protein. The native variant of a truncated polypeptide can be from any organism, including mammalian species such as mouse, rat, rabbit, and human. The engineered polynucleotides and polypeptides described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide or polypeptide. or polypeptide. In some embodiments, a variant of a native polypeptide is a truncated variant of the native polypeptide. As used herein, the term "truncated variant" refers to a protein or polypeptide in which one or more sequences and / or domains of amino acids are missing as compared to the endogenous form of the protein or polypeptide. For example, a truncated variant incorporated into a polypeptide construct described herein may often be missing the sequence of amino acids corresponding to a domain (e.g., an intracellular signaling domain, a transmembrane domain, a ligand-binding domain, etc.) that is present in the endogenous protein. The native variant of a truncated polypeptide can be from any organism, including mammalian species such as mouse, rat, rabbit, and human. The engineered polynucleotides and polypeptides described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide or polypeptide. or domain that is present in the endogenous form of the protein or polypeptide. For example, a truncated variant incorporated into a polypeptide construct described herein may often be missing the sequence of amino acids corresponding to a domain (e.g., an intracellular signaling domain, a transmembrane domain, a ligand-binding domain, etc.) that is present in the endogenous protein. The native variant of a truncated polypeptide can be from any organism, including mammalian species such as mouse, rat, rabbit, and human. The engineered polynucleotides and polypeptides described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide or polypeptide. or polypeptide. For example, a truncated variant incorporated into a polypeptide construct described herein may often be missing the sequence of amino acids corresponding to a domain (e.g., an intracellular signaling domain, a transmembrane domain, a ligand-binding domain, etc.) that is present in the endogenous protein. The native variant of a truncated polypeptide can be from any organism, including mammalian species such as mouse, rat, rabbit, and human. The engineered polynucleotides and polypeptides described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide such as intracellular signaling domain, transmembrane domain, ligand-binding domain etc.). The native variant of a truncated polypeptide can be from any organism, including mammalian species such as mouse, rat, rabbit, and human. The engineered polynucleotides and polypeptides described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide or polypeptide. or polypeptide. The engineered polynucleotides and polypeptides described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide or polypeptide. As used herein, an engineered cell is a cell that has been modified from its native or endogenous state. Examples of engineered cells include cells that have been modified to encode a truncated variant of a native polypeptide or a truncated variant of a native polynucleotide or polypeptide. For example, by transfection of a polynucleotide into a cell), are the cells described herein. as described herein.

[0094] Polypeptide construct As used herein, a polypeptide construct, a polynucleotide encoding the same, an operation cell carrying and / or expressing the polypeptide construct and the polynucleotide, and a method for regulating the activity of the operation cell are disclosed. The operation cells described herein can include immune effector cells engineered to encode and express cytokines, chimeric antigen receptors, and T cell receptors. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell.

[0095] As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell. As used herein, the term "regulating" or "regulation" when referring to an engineered cell or a polypeptide construct to be expressed therein generally refers to the regulation of the activity or amount of the engineered cell after administration to a subject. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell in the subject. In some embodiments, regulating the activity of the engineered cell refers to depleting some of the engineered cells in the subject as a result of administering to the subject an antibody or binding partner that binds to a polypeptide construct expressed on the engineered cells in a certain amount. In some embodiments, regulating the activity of the engineered cell refers to depleting the engineered cell as a result of activating cell death through the binding of an antibody or binding partner to a polypeptide construct described herein that is expressed on or associated with the engineered cells. In some embodiments, regulating the activity of the engineered cell refers to activating the ADCC pathway or the CDC pathway within the engineered cell.

[0096] The polypeptides, polynucleotides, and engineered cells disclosed herein are collectively useful for improving the effectiveness of conventional immunotherapies, and in particular, for improving the effectiveness of adoptive cell therapies. For example, the engineered cells described herein can encode and express a polypeptide construct as a novel cell tag on the cell surface. In some embodiments, the cell tag can function as a cell marker to label, mark, or flag (flag) the cell expressing the cell tag as an engineered cell. In multiple embodiments, when the cell tag is engineered to lack an epitope recognized by an endogenous protein, such a cell tag can function to uniquely identify the engineered cell from other cells in an organism, for example, during adoptive cell immunotherapy. In other examples, the polypeptides, polynucleotides, and engineered cells described herein can be used to minimize or eliminate the toxicity of immunotherapy in a subject when safety is a concern (e.g., due to the potential for cytokine storm). The cell tags described herein can include one or more epitopes that are recognized by an antibody introduced during immunotherapy, thereby inducing a cell mechanism that slows down the therapeutic output and / or reduces the possible side effects of the treatment. In some embodiments, the antibody binds to the epitope and induces antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Thus, the cell tags described herein allow medical practitioners to control the therapeutic output, thereby

[0097] In other examples, the polypeptides, polynucleotides, and engineered cells described herein can be used to minimize or eliminate the toxicity of immunotherapy in a subject when safety is a concern (e.g., due to the potential for cytokine storm). The cell tags described herein can include one or more epitopes that are recognized by an antibody introduced during immunotherapy, thereby inducing a cell mechanism that slows down the therapeutic output and / or reduces the possible side effects of the treatment. In some embodiments, the antibody binds to the epitope and induces antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Thus, the cell tags described herein allow medical practitioners to control the therapeutic output, thereby In some embodiments, the antibody binds to the epitope and induces antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Thus, the cell tags described herein allow medical practitioners to control the therapeutic output, thereby ​​​​​​​​​​​result in depletion markers or “kill tags” specific to engineered cells that enable optimization of treatment efficacy and safety.

[0098] The immunotherapeutic means described herein improve conventional immunotherapies by providing potential for control over adoptive cell therapy interventions. In some embodiments, engineered cells can be sensitized to cell depletion strategies by expressing polypeptide constructs on the surface of the engineered cells that are capable of dimerizing or multimerizing. Such dimerizing polypeptides or multimerizing polypeptides can be used to amplify depletion signals, thereby either rapidly downregulating or eliminating engineered adoptive immunotherapies in a subject that is susceptible to or already experiencing treatment-related side effects. Administration of engineered cells expressing a dimerizing polypeptide construct or a multimerizing polypeptide construct can provide additional control and optimization over interventions by cell depletion. In further embodiments, implementation of the inducible promoter-based “kill switch” (or “suicide switch”) system of the polynucleotide constructs disclosed herein enables control over the timing of expression of specific polypeptide constructs, thereby providing an additional point of control over immunotherapies at both the transcriptional and post-translational levels. In another embodiment, the cell tags described herein can be used to enrich engineered cells that specifically express such cell tags. For example, cell tags can be used to achieve the purity necessary to enable expansion of selected cells for therapeutic purposes.

[0099] Certain engineered cells that express only cell tags can be isolated by concentrating certain engineered cells. If necessary, various methods can be used, such as FAC, purification by column, or magnetic bead-based methods.

[0100] The polypeptide constructs disclosed herein can include one or more domains or specific fragments thereof. Typically, the polypeptide constructs each include a signal peptide sequence, an extracellular domain, a peptide linker, and a transmembrane domain that function to confer the desired specific properties to the polypeptide construct. For example, the polypeptide construct can include a signal peptide that directs the polypeptide construct to the cell surface after translation; a transmembrane domain that anchors the polypeptide domain to the cell; an extracellular portion that can include a truncated variant of a native polypeptide; and a peptide linker that connects the transmembrane domain to the extracellular portion. In some embodiments, the peptide linker functions as an extracellular peptide extension that positions the extracellular portion of the polypeptide within the extracellular matrix, thereby making this portion available for conferring functionality to the cell tag (e.g., presenting an epitope for binding to an antibody or extending it). In certain embodiments, one or more of the above domains may be absent from the polypeptide construct. For example, the polypeptide constructs described herein can be engineered to lack a peptide linker domain. In other embodiments, the polypeptide construct includes one or more domains that are derived from different proteins (i.e., the polypeptide construct is chimeric). ​

[0101] As used herein, when referring to a portion of a polypeptide construct, the term "extracellular" refers to the amino acids of the polypeptide located outside the cell membrane. In some embodiments, the extracellular portion can be referred to as a cell surface polypeptide. Typically, a portion (e.g., the distal portion) of the cell surface polypeptide extends or protrudes distally from the cell membrane of the cell into the extracellular lumen. In some cases, the extending portion binds to an antibody or antigen recognition polypeptide having a structure complementary to and specific for the structure of the extending portion, whereby it is recognized by these. The cell surface polypeptide includes, in addition to polypeptides or fragments thereof naturally found on the cell surface, polypeptides or fragments thereof (e.g., truncated variants of native polypeptides) that are not naturally found on the cell surface. The cell surface polypeptide can include truncated variants of native polypeptides or fragments thereof. Truncated variants can extend into the extracellular lumen, for example, from outside the cell membrane (e.g., followed by or adjacent to a transmembrane domain via a linker). In certain embodiments, the truncated variant is a naturally occurring variation of the polypeptide that has lost amino acids contributing to the intracellular domain and / or transmembrane domain.

[0102] The extracellular portion or cell surface polypeptide can include truncated variants of native polypeptides or fragments thereof. Truncated variants can extend into the extracellular lumen, for example, from outside the cell membrane (e.g., followed by or adjacent to a transmembrane domain via a linker). In certain embodiments, the truncated variant is a naturally occurring variation of the polypeptide that has lost amino acids contributing to the intracellular domain and / or transmembrane domain. Truncated variants can be modified in any way from an endogenous polypeptide so as to provide an extracellular portion of a cell tag. For example, truncated variants typically have an epitope within the extracellular domain that can be recognized by an endogenous protein such as an antigen or receptor. or receptor. It is possible to reduce or modify amino acids that function to include P so that they are eliminated. Typically, by removing amino acids that form an interface with extracellular molecules (e.g., within a cell signaling pathway), a truncated variant can be made non-reactive or immunologically / epitopically silent at the cell surface, and its reactivity or binding to endogenous molecules can also be reduced or minimized. As used herein, any modification to a native polypeptide that removes a native epitope, including truncation of all or part of the extracellular domain, and removal of one or more amino acids that form part of or are post-translationally modified to form part of an epitope, is contemplated. The truncated variants described herein are epitopically silent with respect to endogenous signaling pathways, but the truncated variants can typically be recognized by molecules (e.g., antibodies) that do not contact the cell surface corresponding to the engineered cells described herein, including one or more epitopes. In some embodiments, an antibody or binding partner specific for an epitope of the truncated variant is introduced exogenously (e.g., during adoptive cell immunotherapy using adoptive cells). In this regard, an epitope of the truncated variant that can be recognized by the introduced antibody or fragment thereof can be referred to as a cryptic epitope or a latent epitope. That is, a cell tag incorporated into the cell surface of the engineered cells used in adoptive immunotherapy contains a cryptic epitope that remains dormant until an appropriate molecule is introduced into the subject to induce or activate the cell tag (i.e., via recognition of the epitope).

[0103] ​​​​​​​​​​​​​ be tolerated. Such epitopes do not interfere with the therapeutic output of the engineered cells (i.e., the epitope is silent or maintains quiescence) when the treatment is proceeding as desired, but can provide an inducement to downregulate the output of the cells when for any reason it is necessary to temper the immunotherapy. The present disclosure contemplates the use of any antibody or small molecule capable of recognizing an epitope of a cellular tag so as to suppress the therapeutic output of the engineered cells expressing the tag via such elimination of the cells (e.g., via ADCC or CDC). Non-limiting examples of antibodies that can be used to recognize epitopes of the extracellular domain of a cellular tag (e.g., a truncated variant) include rituximab, cetuximab, gefitinib, erlotinib, afatinib, brigatinib, icotinib, osimertinib, panitumumab, zalutumumab, nimotuzumab, matuzumab, afucosylated, obinutuzumab, ibritumomab tiuxetan, tositumomab, ofatumumab, ocrelizumab, TRU-015 (Trubion), belzutifan (IMMU-106), alemtuzumab, ANT1034, HI186 (Bio Rad), YTH34.5 (Bio Rad), and YTH66 .9HL (Bio-Rad), trastuzumab, and pertuzumab. Another example of a modification that can be made to an endogenous polypeptide to create a truncated variant contemplated herein is typically to remove one or more amino acids that contribute or participate in an intracellular signaling pathway and / or trafficking pathway. In the context of the present disclosure, such removal can result in a truncated variant that is more suitable for use as a cellular tag. For example, removing amino acids that are involved in intracellular signaling can prevent the tag from being recognized by immune cells, while removing amino acids that are involved in trafficking can alter the localization of the tag within the cell. These modifications can be used to engineer cellular tags with desirable properties, such as increased stability, altered immunogenicity, or improved targeting capabilities. In some embodiments, the truncated variant can be designed to exhibit a specific pattern of glycosylation or phosphorylation, which can further modulate its function.

[0104] To create the truncated variants contemplated herein, another example of a modification that can be applied to an endogenous polypeptide is typically to remove one or more amino acids that contribute to or participate in an intracellular signaling pathway and / or trafficking pathway. This can result in a truncated variant that is more suitable for use as a cellular tag. For example, removing amino acids involved in intracellular signaling can prevent the tag from being recognized by immune cells, while removing amino acids involved in trafficking can alter the localization of the tag within the cell. Truncation of an endogenous polypeptide that removes the signaling domain enhances the function of the cell tag as a dormant and inducible cell marker that, in its dormant state, does not interfere with cell function (e.g., within a transplanted cell during adoptive cell therapy). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are

[0105] In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271). In some embodiments, the cell surface polypeptide of the polypeptide construct can include a truncated mutant of a receptor tyrosine kinase. Non-limiting examples include truncated mutants derived from receptors of the EGF receptor family (e.g., a truncated mutant of HER1), the PDGF receptor family, the VEGF receptor family, the insulin receptor family, the FGF receptor family, the Trk receptor family, and the Eph receptor family. In other embodiments, the cell surface polypeptide can include a truncated mutant of a CD protein (e.g., CD20 or CD52), or a truncated mutant of LNFGR (CD271).

[0106] In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are In certain embodiments, the cell surface polypeptide of the cell tag can include a truncated mutant that has been modified to remove the transmembrane domain and the intracellular signaling portion of a native polypeptide or an endogenous polypeptide. Truncation of the transmembrane domain and the intracellular signaling portion liberates the cell surface portion or extracellular portion of the polypeptide from its endogenous context and makes it available for incorporation into chimeric polypeptide constructs that include, for example, a cell surface polypeptide fused (e.g., via a linker) to a transmembrane domain derived from a different native protein. Such chimeric polypeptide constructs are A peptide (e.g., a truncated variant) can be modified to have an altered activity or characteristic as compared to the endogenous form of the polypeptide. In some embodiments, the cell surface polypeptide can dimerize when expressed in a chimeric polypeptide construct.

[0107] The present disclosure contemplates that multiple different polypeptide constructs can be expressed in the same host cell. For example, the cells disclosed herein can express multiple polypeptide constructs that differ in the identity of the cell surface polypeptide and / or transmembrane domain.

[0108] The polypeptide constructs described herein can include a truncated variant of a native polypeptide, a transmembrane domain fused to a cell surface polypeptide, optionally a linker connecting the truncated variant to the transmembrane domain, and a signal peptide that directs the cell tag to the cell surface of the host cell.

[0109] Signal Peptide A signal peptide is an amino acid sequence that is typically located at the N-terminus of a newly synthesized protein or polypeptide and that directs the protein or polypeptide to the cell surface. In some embodiments, the signal peptide directs a polypeptide that is inserted into the cell membrane (e.g., via a transmembrane domain) to the cell surface. In some embodiments, the polypeptide constructs described herein are synthesized with a signal peptide but are then post-translationally processed to cleave the signal peptide such that the mature polypeptide construct lacks the amino acid sequence of the signal peptide. ​​​​​​​​In an embodiment, the signal peptide sequence is not cleaved and remains within the mature polypeptide construct. Survive.

[0110] The present disclosure presents a polypeptide construct comprising any known or unknown signal peptide capable of directing and / or trafficking the polypeptide construct to the cell surface. For example, in some embodiments, the polypeptide construct is GMCSFR α, Ig kappa, immunoglobulin E, CD8α, TVB2 (T21A), CD52, or Also includes a signal sequence corresponding to the signal peptide of the low affinity nerve growth factor receptor (LNGFR, TNFRSF16). α, Ig kappa, immunoglobulin E, CD8α, TVB2 (T21A), CD52, or Also includes a signal sequence corresponding to the signal peptide of the low affinity nerve growth factor receptor (LNGFR, TNFRSF16). Survive.

[0111] In a plurality of embodiments, the signal peptide is at least 70%, 75%, 80%, 85%, 90%, 95% Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 Selected from the list consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13

[0112] Peptide linker The polypeptide constructs described herein are peptides that connect a domain of the polypeptide construct or a fragment thereof to a different domain of the polypeptide construct or a fragment thereof. The polypeptide constructs described herein are peptides that connect a domain of the polypeptide construct or a fragment thereof to a different domain of the polypeptide construct or a fragment thereof. It may include a linker. In some embodiments, the peptide linker connects the transmembrane domain of the polypeptide construct to the cell surface polypeptide of the polypeptide construct (e.g., a truncated variant of a native polypeptide). For example, the polypeptide construct may include a GSG linker (SEQ ID NO: 16), an SGSG linker (SEQ ID NO: 18), a (G4S)3 linker (SEQ ID NO: 20), a (G4S)4 linker (SEQ ID NO: 22), and / or a peptide linker including a Whitlow linker. The transmembrane domain is connected to the cell surface polypeptide of the polypeptide construct (e.g., a truncated variant of a native polypeptide). For example, the polypeptide construct may include a GSG linker (SEQ ID NO: 16), an SGSG linker (SEQ ID NO: 18), a (G4S)3 linker (SEQ ID NO: 20), a (G4S)4 linker (SEQ ID NO: 22), and / or a peptide linker including a Whitlow linker. The transmembrane domain is connected to the cell surface polypeptide of the polypeptide construct (e.g., a truncated variant of a native polypeptide). For example, the polypeptide construct may include a GSG linker (SEQ ID NO: 16), an SGSG linker (SEQ ID NO: 18), a (G4S)3 linker (SEQ ID NO: 20), a (G4S)4 linker (SEQ ID NO: 22), and / or a peptide linker including a Whitlow linker. The transmembrane domain is connected to the cell surface polypeptide of the polypeptide construct (e.g., a truncated variant of a native polypeptide). For example, the polypeptide construct may include a GSG linker (SEQ ID NO: 16), an SGSG linker (SEQ ID NO: 18), a (G4S)3 linker (SEQ ID NO: 20), a (G4S)4 linker (SEQ ID NO: 22), and / or a peptide linker including a Whitlow linker. The transmembrane domain is connected to the cell surface polypeptide of the polypeptide construct (e.g., a truncated variant of a native polypeptide). For example, the polypeptide construct may include a GSG linker (SEQ ID NO: 16), an SGSG linker (SEQ ID NO: 18), a (G4S)3 linker (SEQ ID NO: 20), a (G4S)4 linker (SEQ ID NO: 22), and / or a peptide linker including a Whitlow linker.

[0113] As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface. As used herein, any length or size of peptide linker that connects the transmembrane domain to the cell surface polypeptide (e.g., a truncated variant) is presented. For example, in some embodiments, the peptide linker is sized to maintain the distance between the truncated variant and the transmembrane domain at approximately the same distance as that which occurs between the native non-truncated form of the polypeptide and its endogenous transmembrane domain. In multiple embodiments, the truncated variants described herein are linked to the transmembrane domain via G4S linkers of different sizes (G4S)n [where n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. For example, if two different truncated variants of the same native polypeptide are of different lengths, the shorter truncated variant can be complemented with a larger-sized linker to position both truncated variants at approximately the same distance from the cell surface.

[0114] In certain embodiments, a peptide linker is used to connect domains other than the transmembrane domain. or portions thereof can be linked together. For example, a peptide linker can connect two protein portions of a cell surface polypeptide. In some cases, the cell surface poly peptide can be a chimeric polypeptide and can include truncated variants derived from multiple native polypeptides that can be connected via a peptide linker. An example is a polypeptide construct that includes HER1t in combination with one or more truncated variants of another member of the E GFR family (e.g., HER2, ErbB3, and ErbB4). In other cases, the cell surface polypeptide can be a concatamer of two or more copies of a truncated variant (e.g., a sequence number 123 that includes two copies of a CD20 truncated polypeptide linked via an SG S linker) that is connected via a peptide linker. In multiple embodiments, the peptide linker is encoded by a polynucleotide that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of sequence number 15; sequence number 17; sequence number 1 9; sequence number 21; and sequence number 23. In multiple embodiments, the peptide linker includes an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of sequence number 16; sequence number 18; sequence number 20; sequence number 22; and sequence number 24.

[0115] In multiple embodiments, the peptide linker is encoded by a polynucleotide that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of sequence number 15; sequence number 17; sequence number 1 9; sequence number 21; and sequence number 23. In multiple embodiments, the peptide linker includes an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of sequence number 16; sequence number 18; sequence number 20; sequence number 22; and sequence number 24. In multiple embodiments, the peptide linker includes an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of sequence number 16; sequence number 18; sequence number 20; sequence number 22; and sequence number 24.

[0116] Transmembrane domain The polypeptide constructs described herein can be inserted into the cell membrane and can include transmembrane domains that can anchor the polypeptide construct to the cell surface. The present disclosure presents polypeptide constructs that include one or more of any known or unknown transmembrane domains or fragments thereof. In some embodiments, the transmembrane domain of the polypeptide construct can include a transmembrane domain derived from and / or homologous to one or more native proteins. In some embodiments, the transmembrane domain of the polypeptide construct includes an amino acid sequence corresponding to the transmembrane domain of a single native protein. In some embodiments, the transmembrane domain of the polypeptide construct includes a chimeric transmembrane domain that includes amino acid sequences derived from two or more native proteins. The polypeptide constructs described herein can include transmembrane domains that are single-pass or multi-pass and can anchor the polypeptide construct to the cell surface. CD8α is an example of a protein having a single-pass transmembrane domain. In some embodiments, the polypeptide constructs disclosed herein include a transmembrane domain derived from or homologous to the CD8α protein or a fragment thereof. In some embodiments, the transmembrane domain of the polypeptide construct is encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the transmembrane domain has at least 70 to the amino acid sequence of SEQ ID NO: 34 In some embodiments, the transmembrane domain of the polypeptide construct can include a transmembrane domain derived from and / or homologous to one or more native proteins. In some embodiments, the transmembrane domain of the polypeptide construct includes a transmembrane domain derived from and / or homologous to one or more native proteins. In some embodiments, the transmembrane domain of the polypeptide construct includes an amino acid sequence corresponding to the transmembrane domain of a single native protein. In some embodiments, the transmembrane domain of the polypeptide construct includes an amino acid sequence corresponding to the transmembrane domain of a single native protein. In some embodiments, the transmembrane domain of the polypeptide construct includes a chimeric transmembrane domain that includes amino acid sequences derived from two or more native proteins. In some embodiments, the transmembrane domain of the polypeptide construct includes a chimeric transmembrane domain that includes amino acid sequences derived from two or more native proteins.

[0117] The polypeptide constructs described herein can include transmembrane domains that are single-pass or multi-pass and can anchor the polypeptide construct to the cell surface. CD8α is an example of a protein having a single-pass transmembrane domain. In some embodiments, the polypeptide constructs disclosed herein include a transmembrane domain derived from or homologous to the CD8α protein or a fragment thereof. In some embodiments, the polypeptide constructs disclosed herein include a transmembrane domain derived from or homologous to the CD8α protein or a fragment thereof. In some embodiments, the transmembrane domain of the polypeptide construct includes a transmembrane domain derived from or homologous to the CD8α protein or a fragment thereof. In some embodiments, the transmembrane domain of the polypeptide construct is encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the transmembrane domain of the polypeptide construct is encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the transmembrane domain of the polypeptide construct is encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the transmembrane domain has at least 70 comprising an amino acid sequence having an identity of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

[0118] Examples of multi-pass transmembrane proteins include CD28. In some embodiments, the transmembrane domain of the polypeptide construct may correspond to or include a transmembrane domain derived from the CD28 protein or a fragment thereof, and / or a transmembrane domain homologous thereto. In multiple embodiments the transmembrane domain of the polypeptide construct is encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100 % identity to the nucleotide sequence of SEQ ID NO: 35. In multiple embodiments the transmembrane domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the transmembrane domain is a transmembrane dimerization domain. As used herein a "transmembrane dimerization domain" refers to a transmembrane domain or a fragment thereof that is capable of physically interacting with or "dimerizing" with a second transmembrane domain or a fragment thereof within the cell membrane of a cell. Typically, a transmembrane dimerization domain is included within a first polypeptide construct that dimerizes with a second polypeptide via the transmembrane dimerization domain. By fusing the transmembrane dimerization domain of the first polypeptide at its distal (extracellular-directed) end to a first cell surface polypeptide and fusing the transmembrane dimerization domain of the second polypeptide at its distal end to a second cell surface polypeptide, when

[0119] ​​​​​​​​​​, the physical interaction between the first transmembrane domain and the second transmembrane domain within the cell membrane can result in the dimerization of the first cell surface polypeptide and the second cell surface polypeptide. In some embodiments, the transmembrane domains can multimerize to form trimers, tetramers , or multimers.

[0120] In certain embodiments, the transmembrane dimerization domain induces dimerization of the cell surface polypeptide without the need for an extracellular inducer (e.g., a ligand or an antibody specific for an epitope of the cell surface polypeptide). For example, the transmembrane dimerization domain can spontaneously physically interact or conjugate with a second transmembrane dimerization domain within the cell membrane of the cell to thereby induce dimerization of the cell surface polypeptide. Thus, it will be understood that cells expressing the polypeptide constructs described herein can present dimerized cell surface polypeptides on the cell surface of the cell prior to administration of any extracellular cell surface binding agent, including the antibodies, proteins, ligands, or molecules described herein. Dimerization of the cell surface polypeptide via the transmembrane dimerization domain can enhance the response of the cell when the dimerized cell surface polypeptide contacts and recognizes a ligand or antibody. Thus, cells expressing such polypeptide constructs can be utilized. For example, if the polypeptide construct includes a cell surface polypeptide comprising HER1t, the pair of HER1t cell surface polypeptides can be dimerized prior to or upon contact with a CDC inducer or an ADCC inducer such as cetuximab. As a result of the dimerization conformation of the cetuximab-binding cell surface polypeptide, upon distress (e.g., cytokine storm The dimerization of the cell surface polypeptide via the transmembrane dimerization domain, when the dimerized cell surface polypeptide contacts and recognizes a ligand or antibody, can enhance the response of the cell. Thus, cells expressing such polypeptide constructs can be utilized. For example, if the polypeptide construct includes a cell surface polypeptide comprising HER1t, the pair of HER1t cell surface polypeptides can be dimerized prior to or upon contact with a CDC inducer or an ADCC inducer such as cetuximab. As a result of the dimerization conformation of the cetuximab-binding cell surface polypeptide, upon distress (e.g., cytokine storm ER1t cell surface polypeptide pairs can be dimerized prior to or upon contact with a CDC inducer or an ADCC inducer such as cetuximab. As a result of the dimerization conformation of the cetuximab-binding cell surface polypeptide, upon distress (e.g., cytokine storm induced dimerization of the cetuximab-binding cell surface polypeptide, Administration of the binder in the arm amplifies the cytotoxic effect, thereby increasing the ability to kill cells. In some embodiments, a drug that induces a cellular response within an engineered cell that binds to a cell surface polypeptide (e.g., a dimerized cell surface polypeptide) and expresses the polypeptide construct disclosed herein is exogenously administered and / or endogenously does not exist. The present disclosure presents a transmembrane dimerization domain that facilitates dimerization of any cell surface polypeptide, including truncated variants of native polypeptides, such as HER1t, LNGFRt, CD20t, and CD52t. In some embodiments, the transmembrane dimerization domain forms a covalent linkage with a second transmembrane domain to induce dimerization of the cell surface polypeptide. In some embodiments, the covalent connection is in the form of a disulfide bond formed between cysteine amino acids present in each of the adjacent transmembrane domains. In other embodiments, the transmembrane dimerization domain within the cell membrane forms a non-covalent connection with a second transmembrane domain to induce dimerization of the cell surface polypeptide.

[0121] The polypeptide constructs described herein can have a transmembrane dimerization domain that physically interacts with another transmembrane dimerization domain. In such cases, the first transmembrane dimerization domain and the second transmembrane dimerization domain of each first polypeptide construct and the second polypeptide construct may have the same amino acid sequence (i.e., a homodimer with respect to the transmembrane dimerization domain), or different amino acid sequences (i.e., a heterodimer). heterodimer). In some embodiments, the transmembrane dimerization domain forms a non-covalent connection with a second transmembrane domain to induce dimerization of the cell surface polypeptide.

[0122] The polypeptide constructs described herein can have a transmembrane dimerization domain that physically interacts with another transmembrane dimerization domain. In such cases, the first transmembrane dimerization domain and the second transmembrane dimerization domain of each first polypeptide construct and the second polypeptide construct may have the same amino acid sequence (i.e., a homodimer with respect to the transmembrane dimerization domain), or different amino acid sequences (i.e., a heterodimer). For the transmembrane dimerization domain, it may have the same amino acid sequence (i.e., a homodimer), or different amino acid sequences (i.e., a heterodimer). , with respect to the transmembrane dimerization domain, it may have a heterodimer). In certain embodiments In certain embodiments, each transmembrane dimerization domain of the dimerization polypeptide pair is the first Within the transmembrane dimerization domain and within the second transmembrane dimerization domain, corresponding cyst Contains at least one cysteine residue that mediates the formation of a disulfide bridge between residues .

[0123] The transmembrane domain of the polypeptide construct may correspond to and / or contain an amino acid sequence homologous to the amino acid sequence of glycophorin A, which is a protein. In some embodiments The amino acid sequence of glycophorin A incorporated into the polypeptide constructs described herein Contains the transmembrane domain of glycophorin A or a fragment thereof. In some embodiments The transmembrane domain of the polypeptide construct usually contains one or more amino acids adjacent to the glycophorin A transmembrane domain . In some embodiments, a polypeptide construct containing all or part of the glycophorin A transmembrane domain can dimerize (e.g., homodimerize) with a second polypeptide construct containing all or part of the glycophorin A transmembrane domain . In such cases, the amino acid sequence incorporated from glycophorin A into the polypeptide construct can define the transmembrane dimerization domain. For example The glycophorin A dimerization domain may contain the GXXXG, which is a dimerization motif . .

[0124] In some embodiments, the polypeptide construct contains a transmembrane domain of glycophorin A that includes at least amino acids E91 - R116. In some embodiments, the polypeptide construct The substance contains a transmembrane domain of glycophorin A that includes at least amino acids I92 - I114 In multiple embodiments, the polypeptide construct includes a transmembrane domain corresponding to the glycophorin A domain or a fragment thereof, encoded by a polynucleotide that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 25 and SEQ ID NO: 27 In multiple embodiments, the polypeptide construct includes a transmembrane domain corresponding to the glycophorin A domain or a fragment thereof, having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 26 and SEQ ID NO: 28 In multiple embodiments, the polypeptide construct includes a transmembrane domain corresponding to the glycophorin A domain or a fragment thereof, having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 26 and SEQ ID NO: 28 In multiple embodiments, the polypeptide construct includes a transmembrane domain corresponding to the glycophorin A domain or a fragment thereof, having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 26 and SEQ ID NO: 28 .

[0125] The polypeptide constructs described herein can include a transmembrane domain that is a chimera of amino acid sequences derived from two or more native polypeptides. For example, the transmembrane domain can be linked or fused to an amino acid sequence derived from a second protein and can include an amino acid sequence corresponding to the glycophorin A domain or a portion thereof. In some embodiments, such a chimeric transmembrane domain can dimerize with a second transmembrane domain (e.g., chimeric or non - chimeric), thus resulting in a transmembrane dimerization domain. For example, an amino acid sequence corresponding to the glycophorin A transmembrane domain or a fragment thereof can be fused to an amino acid sequence corresponding to a domain of integrin β3 or a fragment thereof to form a chimeric transmembrane domain of the polypeptide construct ​​​​​​​​​​​​It is. In some embodiments, the polypeptide construct is the amino acid A737 of integrin β3 ~W741 is fused to the transmembrane domain containing amino acids I92~L109 of glycophorin A Including. In multiple embodiments, the polypeptide construct is the nucleotide of SEQ ID NO: 29 At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or Also contains a nucleotide sequence having 100% identity with the polynucleotide Encoded, including a transmembrane domain corresponding to the glycophorin A-integrin β3 chimeric sequence In multiple embodiments, the polypeptide construct is at least At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% Having identity and including a transmembrane domain.

[0126] The transmembrane domain of the polypeptide construct corresponds to the amino acid sequence within the transmembrane domain of the CD3 zeta chain And / or may include an amino acid sequence homologous to this. In some embodiments Is, a polypeptide construct containing the transmembrane domain of the CD3 zeta chain or a fragment thereof is CD 3 Can dimerize with a second polypeptide construct containing the transmembrane domain of the zeta chain or a fragment thereof (For example, homodimerize). In such a case, the amino acid sequence corresponding to the CD3 Zeta chain transmembrane domain can define a transmembrane dimerization domain In some embodiments, the polypeptide construct is relative to the nucleotide sequence of SEQ ID NO: 31 At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 1 00% identity and contains a transmembrane domain encoded by a polynucleotide having identity In other embodiments, the transmembrane domain is the nucleotide of SEQ ID NO: 31 Encoded by a polynucleotide comprising a D array. In a plurality of embodiments, the polypeptide construct comprises a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 32 .

[0127] In other embodiments, the transmembrane domain of the polynucleotide construct herein is a transmembrane domain corresponding to or homologous to the transmembrane domain or a fragment thereof derived from the protein CTLA4 (cytotoxic T lymphocyte protein 4) and / or LNGFR (TNFRSF16), and may comprise an amino acid sequence identical or homologous thereto. For example, the polypeptide construct may comprise a transmembrane domain encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 37 and SEQ ID NO: 39. In a plurality of embodiments, the polypeptide construct comprises a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 38 and SEQ ID NO: 40 . .

[0128] Truncated variant The polypeptide constructs described herein may include a cell surface polypeptide linked to a transmembrane domain. In certain embodiments, the cell surface polypeptide comprises a truncated variant of a native polypeptide. As used herein, a variety of native polypeptides ​​​​​​​​​, a precursor or presented as a substrate. Each natural polypeptide precursor gives rise to a number of possible truncated variants for use in the polypeptide constructs described herein, and can be further subjected to a plurality of different truncations.

[0129] Examples of precursors include epidermal growth factor receptor (EGFR or HER1) isoform precursors (e.g., SEQ ID NO: 50); receptor tyrosine protein kinase ErbB2 (HER2) isoform precursors (e.g., SEQ ID NO: 51); receptor tyrosine protein kinase E rbB3 (HER3) isoform 1 precursor (e.g., SEQ ID NO: 52), receptor tyrosine protein kinase ErbB4 (HER4) isoform JM-a / CVT-1 precursor (e.g., SEQ ID NO: 53), receptor tyrosine protein kinase ErbB4 (HER4 ) isoform JM-b isoform X7 (e.g., SEQ ID NO: 54), CD20 precursor (e.g., SEQ ID NO: 108), CD52 precursor, and LNGFR precursor (e.g., SEQ ID NO: 154).

[0130] In certain embodiments, the cell surface polypeptide comprises a truncated HER1 polypeptide (referred to herein as HER1t or EGFRt). Native HER1 comprises an extracellular region containing domains I, II, III, and IV, a transmembrane domain, and an intracellular tyrosine kinase and regulatory region. Certain antibodies capable of inducing ADCC (e.g., panitumumab and cetuximab) are known to bind to domains I II of endogenous HER1.

[0131] As used herein, a polypeptide construct is presented that includes a HER1 polypeptide in which any amino acid, domain, or fragment of endogenous HER1 has been truncated. As used herein a HER1 polypeptide may include a HER1t polypeptide. In some embodiments the HER1 polypeptide consists of, or consists essentially of a HER1t polypeptide. In other embodiments, the HER1 polypeptide may include a HER1t polypeptide in addition to other HER1 domains (e.g., the HER1 transmembrane domain). In multiple embodiments the HER1 polypeptide may lack an intracellular domain or fragment thereof that is normally found within HER1 and that includes a tyrosine kinase domain and a regulatory region. In some embodiments the HER1 polypeptide may lack a transmembrane domain or fragment thereof that is normally found within HER1. In some embodiments, the HER1 polypeptide may lack all or a portion of an extracellular domain or fragment thereof that is normally found within HER1 and that includes domains I, II, and IV of HER1. In some embodiments, the cell surface polypeptide includes a HER1t polypeptide that lacks domain III that is normally found within HER1. In some embodiments, the HER1t polypeptide consists of, or consists essentially of all or a portion of domain III of the endogenous HER1 protein. In some embodiments, the HER1t polypeptide consists of, or consists essentially of all of domain III of the endogenous HER1 protein. In some embodiments, the HER1 polypeptide may lack all or a portion of domains I, II, and IV that are normally found within HER1. In some embodiments, the HER1 polypeptide may lack all or a portion of an extracellular domain or fragment thereof that is normally found within HER1 and that includes domains I, II, and IV of HER1.

[0132] In some embodiments, the cell surface polypeptide includes a HER1t polypeptide that lacks domain III that is normally found within HER1. In some embodiments, the HER1t polypeptide consists of, or consists essentially of all or a portion of domain III of the endogenous HER1 protein. In some embodiments, the HER1t polypeptide consists of, or consists essentially of all of domain III of the endogenous HER1 protein. In some embodiments, the HER1t polypeptide consists of, or consists essentially of all of domain III of the endogenous HER1 protein. In certain embodiments, the HER1t domain III incorporated into the cell surface polypeptide is , a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 199 and is encoded by a polynucleotide. In certain embodiments, the HER1t domain III incorporated into the cell surface polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 200.

[0133] In some embodiments, the HER1t polypeptide incorporated into the cell surface polypeptide comprises the domain IV of endogenous HER1 or a fragment thereof. The endogenous HER1 domain IV can be encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 201. In certain embodiments, the endogenous HER1t domain IV is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 202. In other embodiments the HER1t polypeptide incorporated into the cell surface polypeptide can comprise a truncated form of domain IV. The HER1t truncated form of domain IV can comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of the native HER1, and can include up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% truncation. In certain embodiments, the cell surface polypeptide The HER1t truncated domain IV incorporated into chid has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, and SEQ ID NO: 209.

[0134] The polypeptide constructs described herein may include a HER1t polypeptide comprising HER1 domains III and IV. In certain embodiments, the HER1t polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 210 and comprises HER1 domains I II and domain IV.

[0135] The polypeptide constructs described herein may include a HER1t polypeptide comprising domain III of HER1 and a fragment of domain IV. In certain embodiments, the HER1t polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, and SEQ ID NO: 217.

[0136] Native HER1 also contains multiple disulfide bond pairs within domain IV. In some embodiments, the HER1t truncated domain IV may include a truncation at a position that preserves the disulfide bond pairs. In further embodiments, as described herein ​ The HER1t variants to be loaded are linked to the transmembrane domain via G4S linkers (G4S)n of different sizes [in the sequence, n = 0, 1, 2, 3, 4, 5] (SEQ ID NO: 222) to maintain the "natural" distance between HER1t and the transmembrane protein. In further embodiments the linker can further remove the 7-residue linker between domain IV and the EGFR transmembrane domain, as it plays a role in the dimerization of the EGFR receptor that results in the activation of the EGF ligand.

[0137] The HER1t polypeptide incorporated into the cell surface polypeptide as described herein may contain epitopes that can be recognized by an exogenously introduced binding partner or antibody. In some embodiments, the HER1t polypeptide may incorporate a cetuximab-binding domain to facilitate the targeted depletion of cells expressing the polypeptide constructs described herein. Depletion may result from cell death arising from, for example, CDC and / or ADC C. Non-limiting examples of molecules that can bind to and / or recognize epitopes on cell surface polypeptides containing HER1t include cetuximab, gefitinib, erlotinib, afatinib, brigatinib, icotinib, osimertinib, panitumumab, zalutumumab, nimotuzumab, and matuzumab. In various embodiments, the antibody can be a monoclonal antibody, scFv, scFab, diabody, or camelid antibody. In another embodiment, the antibody can be conjugated to a drug or toxin.

[0138] ​​​​​​​​​​The cell surface polypeptides incorporated into the polypeptide constructs described herein can include truncated variants of multiple different native polypeptides. For example, the cell surface po lypeptide can include multiple truncated polypeptides derived from the EGFR family, including polypeptide chimeras. Figure 4B illustrates an embodiment of a polypeptide construct that confers functionality to a cell tag, in which case the cell surface polypeptide is HER / EGFR family member (HER(m)) domain III and HER / E GFR family member (HER(n)) domain IV or a fragment thereof. As used herein, any combination of extracellular domains derived from two or more of EGFR / HER1, HER2, ErbB3, and Er bB4 is presented, including chimeric cell surface polypeptides. Figure 4C depicts a specific case where domain III is derived from EGF R / HER1. The advantage of such chimeric cell surface polypeptides is that each individual truncation has a tendency to bind to endogenous molecules while preserving epitopes that can be recognized by exogenously introduced antibodies during adoptive cell therapy. That is, the epitopes of each native polypeptide can be removed. For example, if the chimeric cell surface po lypeptide includes domain III derived from EGFR and domain IV derived from HER2, the engineered cells expressing the polypeptide can be sensitive to both cetuximab (E GFR recognizes domain III) and trastuzumab (HER2 recognizes domain IV). Thus, the chimeric cell surface polypeptides described herein are sensitive to multiple antibiotics / binding partners. For example, when the chimeric cell surface polypeptide includes domain III derived from EGFR and domain IV derived from HER2, the engineered cells expressing the polypeptide can be sensitive to both cetuximab (E GFR recognizes domain III) and trastuzumab (HER2 recognizes domain IV). Thus, the chimeric cell surface polypeptides described herein are sensitive to multiple antibiotics / binding partners. Providing a binding site, a further mechanism for controlling the behavior of immune cells during immunotherapy is provided. For example, in the case of a subject suffering from side effects during adoptive cell therapy, targeting an epitope in one of the truncated variants within the chimeric cell surface polypeptide in a situation where the administered antibiotic (e.g., cetuximab) does not respond, a different antibody (e.g., , trastuzumab) can be administered to the subject to target the same engineered cells via a different epitope on a different truncated variant of the chimeric cell surface polypeptide . In some embodiments, the chimeric cell surface polypeptide can be fused with a transmembrane domain homologous to an EGFR family member . For example, the transmembrane domain can correspond to a transmembrane domain derived from EGFR / HER1, HER2, ErbB3, or ErbB4

[0139] . In other embodiments, the transmembrane domain can be homologous to a non-EGFR transmembrane domain, including transmembrane domains corresponding to SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40 . In some embodiments, the chimeric cell surface polypeptide can include a chimera of a HER1t polypeptide and a truncated HER2 (HER2t) polypeptide or a fragment thereof . For example, the polypeptide construct can include a HER1t / EGF Rt polypeptide containing HER1 domain III, a HER2t polypeptide containing HER2 domain IV, and a HER2 transmembrane domain . In one embodiment, it can be fused to the HER2 transmembrane domain

[0140] . ​The EGFR-HER2 chimeric cell surface polypeptides are represented by SEQ ID NO: 88 and SEQ ID NO: 92. (Delta 16), , 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity In one embodiment, the nucleic acid sequence of the present invention can be encoded by a polynucleotide comprising the nucleotide sequence of H. The EGFR-HER2 chimeric cell surface polypeptide fused to the ER2 transmembrane domain , an amino acid sequence selected from the group consisting of SEQ ID NO:89 and SEQ ID NO:93 (Delta 16) At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or or has 100% identity.

[0141] In some embodiments, the chimeric cell surface polypeptide comprises a HER1t polypeptide and a t Chimeras with truncated ErbB3 (ErbB3t) polypeptide or fragments thereof For example, the polypeptide construct may be a HER1t / E polypeptide comprising HER1 domain III. a GFRt polypeptide, an ErbB3t polypeptide comprising ErbB3 domain IV, In one embodiment, the ErbB3 transmembrane domain The EGFR-ErbB3 chimeric cell surface polypeptide fused to the nucleic acid sequence of SEQ ID NO: 96. At least 70%, 75%, 80%, 85%, 90%, 95% of the nucleotide sequence , 99%, or 100% identity to a polynucleotide having a nucleotide sequence In one embodiment, the ErbB3 gene can be encoded by an EGFR fused to an ErbB3 transmembrane domain. The FR-ErbB3 chimeric cell surface polypeptide has the amino acid sequence of SEQ ID NO:97 , at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% has % identity.

[0142] In some embodiments, the chimeric cell surface polypeptide comprises a chimera of a HER1t polypeptide and a truncated ErbB4 (ErbB4t) polypeptide or a fragment thereof. For example, the polypeptide construct may comprise a HER1t / EGFRt polypeptide comprising domain III, an ErbB4t polypeptide comprising ErbB4 domain IV, and an ErbB 4 transmembrane domain. In some embodiments, the ErbB4t polypeptide may comprise an ErbB4 JM-a extracellular membrane proximal domain encoded by an alternative transcript of ErbB4 JM-a. In some embodiments, the ErbB4t polypeptide may comprise an ErbB4 JM-b extracellular membrane proximal domain encoded by an alternative transcript of ErbB4 JM-b. In certain embodiments, the chimeric cell surface polypeptide fused to the ErbB4 transmembrane domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to a nucleotide sequence encoding a nucleotide sequence selected from the list consisting of SEQ ID NO: 100 (EGFR-ErbB4 (JM-a)) and SEQ ID NO: 104 (EGFR-ErbB4 (JM-b)). In certain embodiments, the EGFR-ErbB4 chimeric cell surface polypeptide fused to the ErbB4 transmembrane domain is at least 70%, 75%, 80%, 85%, 90% 95%, 99% or 100% identical to an amino acid sequence selected from the list consisting of SEQ ID NO: 101 (EGFR-ErbB4 (JM -a)) and SEQ ID NO: 105 (EGFR-ErbB4 (JM-b)). encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 100 (EGFR-ErbB4 (JM-a)) and SEQ ID NO: 104 (EGFR-ErbB4 (JM-b)). In certain embodiments, the EGFR-ErbB4 chimeric cell surface polypeptide fused to the ErbB4 transmembrane domain is at least 70%, 75%, 80%, 85%, 90% 95%, 99% or 100% identical to an amino acid sequence selected from the list consisting of SEQ ID NO: 101 (EGFR-ErbB4 (JM -a)) and SEQ ID NO: 105 (EGFR-ErbB4 (JM-b)). ​​

[0143] The polypeptide constructs described herein include a signal peptide, a cell surface polypeptide comprising a HER1t polypeptide (e.g., comprising only HER1t or a chimeric polypeptide comprising HER1t), a transmembrane domain, and optionally, a linker, in any combination. For example, the polypeptide construct may be a cell surface polypeptide comprising a HER1t polypeptide or a chimeric polypeptide comprising HER1t, which is linked or fused via a linker (e.g., one or more (e.g., 1 to 4) copies of G4S (SEQ ID NO: 221)) to a derivative or fragment of the CD28 transmembrane domain. Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 56 (HER1t1); SEQ ID NO: 58 (HER1t2); SEQ ID NO: 60 (HER1t3); SEQ ID NO: 62 (HER1t4); SEQ ID NO: 64 (HER1t5); SEQ ID NO: 66 (HER1t6); SEQ ID NO: 68 (HER1t7); SEQ ID NO: 72 (HER1t8); SEQ ID NO: 76 (HER1t9); SEQ ID NO: 80 (HER1t10); and SEQ ID NO: 84 (HER1t11). Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 55 (HER1t); SEQ ID NO: 57 (HER1t1); SEQ ID NO: 59 (HER1t2); SEQ ID NO: 61 (HER1 t3); SEQ ID NO: 63 (HER1t4); SEQ ID NO: 65 (HER1t5); SEQ ID NO: 67 (HER1t6); SEQ ID NO: 69 (HER1t7); SEQ ID NO: 73 (HER1t8); SEQ ID NO: 77 (HER1t9); SEQ ID NO: 81 (HER1t10); and SEQ ID NO: 85 (HER1t11). Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 55 (HER1t); SEQ ID NO: 57 (HER1t1); SEQ ID NO: 59 (HER1t2); SEQ ID NO: 61 (HER1 t3); SEQ ID NO: 63 (HER1t4); SEQ ID NO: 65 (HER1t5); SEQ ID NO: 67 (HER1t6); SEQ ID NO: 69 (HER1t7); SEQ ID NO: 73 (HER1t8); SEQ ID NO: 77 (HER1t9); SEQ ID NO: 81 (HER1t10); and SEQ ID NO: 85 (HER1t11). (HER1t1); SEQ ID NO: 58 (HER1t2); SEQ ID NO: 60 (HER1t3); SEQ ID NO: 62 (HER1t4); SEQ ID NO: 64 (HER1t5); SEQ ID NO: 66 (HER1t6); SEQ ID NO: 68 (HER1t7); SEQ ID NO: 72 (HER1t8); SEQ ID NO: 76 (HER1t9); SEQ ID NO: 80 (HER1t10); and SEQ ID NO: 84 (HER1t11). Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 55 (HER1t); SEQ ID NO: 57 (HER1t1); SEQ ID NO: 59 (HER1t2); SEQ ID NO: 61 (HER1 (HER1t9); SEQ ID NO: 80 (HER1t10); and SEQ ID NO: 84 (HER1t11). Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 55 (HER1t); SEQ ID NO: 57 (HER1t1); SEQ ID NO: 59 (HER1t2); SEQ ID NO: 61 (HER1 t3); SEQ ID NO: 63 (HER1t4); SEQ ID NO: 65 (HER1t5); SEQ ID NO: 67 (HER1t6); SEQ ID NO: 69 (HER1t7); SEQ ID NO: 73 (HER1t8); SEQ ID NO: 77 (HER1t9); SEQ ID NO: 81 (HER1t10); and SEQ ID NO: 85 (HER1t11). Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 55 (HER1t); SEQ ID NO: 57 (HER1t1); SEQ ID NO: 59 (HER1t2); SEQ ID NO: 61 (HER1 t3); SEQ ID NO: 63 (HER1t4); SEQ ID NO: 65 (HER1t5); SEQ ID NO: 67 (HER1t6); SEQ ID NO: 69 (HER1t7); SEQ ID NO: 73 (HER1t8); SEQ ID NO: 77 (HER1t9); SEQ ID NO: 81 (HER1t10); and SEQ ID NO: 85 (HER1t11). Some embodiments include a cell surface polypeptide comprising a HER1t polypeptide encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 55 (HER1t); SEQ ID NO: 57 (HER1t1); SEQ ID NO: 59 (HER1t2); SEQ ID NO: 61 (HER1 t3); SEQ ID NO: 63 (HER1t4); SEQ ID NO: 65 (HER1t5); SEQ ID NO: 67 (HER1t6); SEQ ID NO: 69 (HER1t7); SEQ ID NO: 73 (HER1t8); SEQ ID NO: 77 (HER1t9); SEQ ID NO: 81 (HER1t10); and SEQ ID NO: 85 (HER1t11). (HER1t6); SEQ ID NO: 69 (HER1t7); SEQ ID NO: 73 (HER1t8); and SEQ ID NO: 77 (HER1t9); SEQ ID NO: 81 (HER1t10); and SEQ ID NO: 85 (HER1t11), an amino acid sequence having at least 70 %, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity, may include a HER1t polypeptide.

[0144] In some embodiments, the polypeptide construct is a cell surface polypeptide including a signal peptide, a chimeric polypeptide including a HER1t polypeptide or a HER1t polypeptide, a transmembrane domain including a transmembrane dimerization domain, and a linker connecting the transmembrane domain and the cell surface polypeptide. For example, the polypeptide construct may include an Ig kappa signal peptide, a specific truncated variant of HER1, a linker (e.g., ( G4S)4 (SEQ ID NO: 22)), and a transmembrane dimerization domain (e.g., a transmembrane domain including I92 - I114 of glycophorin A or a transmembrane domain derived from CD3 zeta). In multiple embodiments, a polypeptide construct including a HER1t polypeptide and a transmembrane dimerization domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 70 (glycophorin A; HER1t8); SEQ ID NO: 74 (glycophorin A; HER1t9); SEQ ID NO: 78 (glycophorin A; HER1t10); In some embodiments, the HER1t polypeptide and the transmembrane dimerization domain are encoded by The polypeptide construct comprising the main sequence is SEQ ID NO: 71 (glycophorin A; HER1t8). ; SEQ ID NO: 75 (Glycophorin A; HER1t9); SEQ ID NO: 79 (Glycophorin A; H ER1t10); and SEQ ID NO: 83 (CD3 zeta; HER1t11) At least 70%, 75%, 80%, 85%, 90%, 100%, 15 ... It includes amino acid sequences with 0%, 95%, 99% or 100% identity.

[0145] In another embodiment, the polypeptide construct comprises a polypeptide linked to a HER2 transmembrane domain. It may include cell surface polypeptides, including HER1t-HER2t chimeras. The HER1t-HER2t chimera and the transmembrane domain are then administered to cells. Further linking to a signal peptide (e.g., GMCSFRα) that directs it to the surface In one embodiment, the polypeptide construct can be SEQ ID NO: 86 and SEQ ID NO: 90(delta16)と5′-nucleotide sequence selected from the list consisting of at least one of the following: 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity In one embodiment, the polypeptide construct is encoded by a polynucleotide having the formula: is an amine selected from the list consisting of SEQ ID NO:87 and SEQ ID NO:91 (Delta 16) At least 70%, 75%, 80%, 85%, 90%, 95%, 99% %, or 100% amino acid sequence identity.

[0146] In another example, the polypeptide construct comprises a HE It may include a cell surface polypeptide containing an R1t-ErbB3t chimera. In certain embodiments a HER1t-ErbB3t chimera and a transmembrane domain are further connected to a signal peptide (e.g., GMCSFRα) that directs the polypeptide construct to the cell surface. In certain embodiments, the polypeptide construct is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 94 and is encoded by a polynucleotide having such identity. In certain embodiments, the poly peptide construct has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 95.

[0147] In yet another example, the polypeptide construct may include a cell surface polypeptide containing a HER1t-ErbB4t chimera linked to an ErbB4 transmembrane domain. In certain embodiments, a HER1t-ErbB4t chimera and a transmembrane domain are further connected to a signal peptide (e.g., GMCSFRα) that directs the polypeptide construct to the cell surface. In certain embodiments, the polypeptide construct is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from the list consisting of SEQ ID NO: 98 (JM-a variant ) and SEQ ID NO: 102 (JM-b variant) and is encoded by a polynucleotide having such identity. In certain embodiments, the polypeptide construct is at least 70% identical to an amino acid sequence selected from the list consisting of SEQ ID NO: 99 , having an identity of 75%, 80%, 85%, 90%, 95%, 99%, or 100% has an amino acid sequence.

[0148] A polypeptide construct comprising a HER1t polypeptide can include any signal peptide capable of directing the polypeptide construct to the cell surface. For example, a cell surface polypeptide comprising a HER1 t polypeptide (e.g., an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 1 00% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 200, SEQ ID NO: 210 , SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215 , SEQ ID NO: 216, and SEQ ID NO: 217) can be fused to a signal peptide having at least 70%, 75%, 8 0%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GMCSFRα), SEQ ID NO: 4 (Ig kappa), SEQ ID NO: 6 (immunoglobulin E), SEQ ID NO: 8 (CD8α), SEQ NO: 10 (TVB2), SEQ ID NO: 12 (CD52), or SEQ ID NO: 14 (LNFGR )). A polypeptide construct comprising a HER1t polypeptide can include any transmembrane domain, including a transmembrane domain that does not include a dimerization domain. For example, a cell surface polypeptide comprising a HER1t polypeptide (e.g., an amino acid sequence having at least 70%, 75%, 8 0%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 200, SEQ ID NO: 210, SEQ ID NO: 2

[0149] 11, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 2 16, and SEQ ID NO: 217) can be fused to a signal peptide having at least 70%, 75%, 8 0%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 200, SEQ ID NO: 210, SEQ ID NO: 2 11, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 2 16, and SEQ ID NO: 217) can include any transmembrane domain, including a transmembrane domain that does not include a dimerization domain. For example, a cell surface polypeptide comprising a HER1t polypeptide (e.g., an amino acid sequence having at least with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity (including an amino acid sequence having such identity) is linked to a transmembrane domain selected from the list consisting of SEQ ID NO: 26 (glycophorin A E91~R116 ), SEQ ID NO: 28 (glycophorin A I92~I114), SEQ ID NO: 30 (glycophorin A (I92~L109). integrin β3 (A737~W741), SEQ ID NO: 32 (C D3 zeta chain), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTLA4) and SEQ ID NO: 40 (LNGFR), with at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence. The polypeptide construct containing the HER1t polypeptide can contain any peptide linker (

[0150] or, in some embodiments, may not contain a peptide linker). For example, a cell surface polypeptide containing HER 1t (e.g., an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 200, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, and SEQ ID NO: 217), to an amino acid sequence selected from the list consisting of SEQ ID NO: 16 (GSG), SEQ ID NO: 18 (SGS G), SEQ ID NO: 20 ((G4S)3), SEQ ID NO: 22 ((G4S)4), and SEQ ID NO: 2 4 (Whitlow), with at least 7 0%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity. 0%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity. It can be fused with a peptide linker containing the amino acid sequence to be used.

[0151] Truncated CD polypeptides can be incorporated into cell surface polypeptides. For example , the cell surface polypeptide can include a truncated CD20 polypeptide (referred to herein as CD 20t). The native CD20 polypeptide is a multi-pass transmembrane protein encoded by the four-pass transmembrane domain subfamily A member 1 (MS4A1) gene. In certain embodiments, full-length CD20 is encoded by a polynucleotide containing the nucleotide sequence of SEQ ID NO: 106, and the full-length CD20 amino acid sequence can correspond to the amino acid sequence of SEQ ID NO: 107. In some embodiments, CD20 includes four transmembrane domain transmembrane portions encompassing amino acids 57-78, 85-105, 121-141, and 189-209. In some embodiments, CD20 includes two extracellular domains encompassing amino acids 79-8 4 and 142-188. In some embodiments , CD20 includes three cytoplasmic domains encompassing amino acids 1-56, 106-120, and 210-297.

[0152] As used herein, polypeptide constructs are presented that include a CD20 polypeptide in which any amino acid, domain, or fragment of endogenous CD20 has been truncated. As used herein , the CD20 polypeptide can include a CD20t polypeptide. In some embodiments , the CD20 polypeptide consists of or consists essentially of a CD20t polypeptide. In other embodiments, the CD20 polypeptide is another CD20 domain or a portion thereof In addition to the moiety (e.g., the CD20 transmembrane domain and / or cytoplasmic domain), it may include a CD2 0t polypeptide. For example, the CD20 polypeptide may truncate the intracellular cytoplasmic (e.g., , signal transduction) domain or a portion thereof, the transmembrane (e.g., helix) domain or a portion thereof, and / or the extracellular domain or a portion thereof. In some embodiments, the CD20 polypeptide may be missing multiple domains or multiple portions of a domain as compared to the wild-type polypeptide. In certain embodiments, the CD20 polypeptide is M1-E263 (SEQ ID NO: 109; CD20t1), M117-N214 (SEQ ID NO: 111 (CD20t2 )) of endogenous CD20, M1-N214 (SEQ ID NO: 115; CD20t4) of endogenous CD20, V82-N214 (SEQ ID NO: 117; CD20t5) of endogenous CD20, or V8 2-I186 (SEQ ID NO: 119, CD20t6) of endogenous CD20.

[0153] In certain embodiments, the CD20t polypeptide may include an extracellular domain or a fragment thereof. In certain embodiments, the CD20t polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220. In some embodiments, the CD20t polypeptide can be linked to a transmembrane domain or a fragment thereof. In certain embodiments, the polypeptide construct includes a CD20t polypeptide linked to a CD20 transmembrane domain. In some embodiments, the CD A polypeptide construct comprising a CD20t polypeptide linked to a transmembrane domain is , SEQ ID NO: 108 (CD20t1 encoding M1 to E263); SEQ ID NO: 110 (M1 17 to N214 encoding CD20t2); SEQ ID NO: 114 (encoding M1 to N214 CD20t4); SEQ ID NO: 116 (CD20t5 encoding V82 to N214); SEQ ID NO: 118 (CD20t6 encoding V82 to I186); SEQ ID NO: 132 (M1 to A54 and C111 to P297 encoding CD20t13); SEQ ID NO: 134 (M 1 to A54 and C111 to E281 encoding CD20t14); SEQ ID NO: 136 ( M1 to A54 and C111 to E263 encoding CD20t15); SEQ ID NO: 138 (M1 to A54 and C111 to V228 encoding CD20t16); and SEQ ID NO : 140 (CD20t17 encoding M1 to V8 and C111 to P297) consisting of At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the List of nucleotide sequences encoded by a polynucleotide having. In certain embodiments, the CD20 transmembrane domain A polypeptide construct comprising a CD20t polypeptide linked to is SEQ ID NO: 109 ( CD20t1: M1 to E263); SEQ ID NO: 111 (CD20t2: M117 to N214 ); SEQ ID NO: 115 (CD20t4: M1 to N214); SEQ ID NO: 117 (CD20t5 : V82 to N214); SEQ ID NO: 119 (CD20t6: V82 to I186); SEQ ID NO : 133 (CD20t13: M1 to A54 and C111 to P297); SEQ ID NO: 135 ( CD20t14: M1 to A54 and C111 to E281); SEQ ID NO: 137 (CD20 t15: M1 to A54 and C111 to E263); SEQ ID NO: 139 (CD20t16: M1 to A54 and C111 to V228); and SEQ ID NO: 141 (CD20t17: M 1 to V8 and C111 to P297) selected from the list consisting of amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 1 00% identity may be included.

[0154] In some embodiments, the CD20t polypeptide is described in Philip et al., (2014), “A highl y compact epitope-based marker / suicide gene for easier and safer T-cell therapy, ” Blood: 124: 1277-1287, and may retain at least one copy of the rituximab-binding mimotope of endogenous CD20. In some embodiments, one or more copies of the rituximab-binding epitope of CD 20t can be fused to one or more amino acid sequences derived from CD34, such as the amino-terminal 40 amino acids of CD34, to facilitate the binding of an anti-CD34 monoclonal antibody. In some embodiments, the CD 20t polypeptide may have a plurality of domains removed, or portions of a plurality of domains removed. The cell surface polypeptide incorporating the CD20t polypeptide may include an epitope that can be recognized by an exogenously introduced antibody or binding partner. For example, the CD20t polypeptide contained within the cell surface po lypeptide may be the polypeptide described herein.

[0155] The cell surface polypeptide incorporating the CD20t polypeptide may include an epitope that can be recognized by an exogenously introduced antibody or binding partner. For example, the CD20t polypeptide contained within the cell surface po lypeptide may be the polypeptide described herein. lypeptide may be the polypeptide described herein. ​To facilitate targeted depletion of cells expressing the construct (e.g., via CDC and / or antibody-dependent cell-mediated cytotoxicity ADCC), a rituximab binding domain can be incorporated. Non-limiting examples of antibodies that can be used to bind to the epitope of a cell surface polypeptide comprising a CD20t polypeptide are rituximab, hOU BM3 / 6, afucosumab, bronetumab, obinutuzumab, ibritumomab tiuxetan, tositumomab, ofatumumab, ocrelizumab, TRU-0 15 (Trubion), and belzutuzumab (IMMU-106). In various embodiments the antibody can be a monoclonal antibody, scFv, scFab, diabody, or can be a camelid antibody. In other embodiments, the antibody can be conjugated to a drug or toxin. In some embodiments, the CD20t polypeptide comprises an epitope found within the extracellular domain of endogenous CD20. In some embodiments, the epitope of the CD 20t polypeptide comprises amino acids 170-185 of endogenous CD20. In some embodiments, the epitope of the CD20t polypeptide comprises amino acids 170-173 and 182-185 of endogenous CD20.

[0156] In further embodiments, the cell surface polypeptide comprising the CD20t polypeptide is chimeric with respect to one or more additional polypeptide truncation mutants. For example the chimeric cell surface polypeptide can comprise a CD20t polypeptide and a truncated CD8α (CD8αt) polypeptide.

[0157] The cell surface polypeptide may contain multiple CD20t-linked sequences in combination as concatamers. For example, the cell surface polypeptide may further comprise a repeat of the same CD20 t amino acid sequence, and may contain different CD20t variants linked together. In some embodiments, the CD20t amino acid sequence is In one embodiment, the linkers are linked together by a peptide linker. G4S linker (SEQ ID NO: 223) was used to and further comprising a cell surface polypeptide linked to the CD28 transmembrane domain. Repeated CD20 amino acid sequences within a surface polypeptide can be linked together (For example, SEQ ID NO:123 (SGS linker) and SEQ ID NO:129 (SG4S linker) (See SEQ ID NO:223).

[0158] The polypeptide constructs described herein comprise a signal peptide and a CD20t polypeptide. and a peptide (e.g., a CD20t polypeptide alone or a CD20t chimeric polypeptide). Any combination of a cell surface polypeptide comprising a transmembrane domain and, optionally, a linker. For example, the polypeptide construct may comprise a cell surface polypeptide comprising a CD20t polypeptide. The SG4S linker (sequence number SG4S) connects or fuses the polypeptide to the transmembrane domain. In certain embodiments, the transmembrane domain may comprise a CD20, C D28, or a transmembrane domain or fragment thereof derived from CD8α, or In one embodiment, the polypeptide construct comprising CD20t has SEQ ID NO: No. 112 (CD20t (K142-S188) and CD8α (I183-T203) membrane A polynucleotide encoding a transmembrane domain CD20t3); SEQ ID NO: 120 (CD20t(P160~Q 187), SG4S linker (SEQ ID NO: 223), and CD28(I96~D172) A polynucleotide encoding a transmembrane domain CD20t7); SEQ ID NO: 122 (CD20t concatamer (P160~Q187 separated by SGS linker), SG4S linker (SEQ ID NO 223), and CD28(I96~D172) A polynucleotide encoding a transmembrane domain CD20t 8); SEQ ID NO: 124 (CD20t(P160~Q187), SG4S linker (SEQ ID num ber 223), and CD8α(P120~V201) A polynucleotide encoding a transmembrane domain CD2 0t9); SEQ ID NO: 126 (CD20t(C167~C183), SG4S linker (SEQ uence number 223), and CD28(I96~D172) A polynucleotide encoding a transmembrane domain CD 20t10; SEQ ID NO: 128 (CD20 concatamer (SG4S linker (SEQ ID number 22 3) separated by C167~C183), SG4S linker (SEQ ID NO: 223), and o and CD28(I96~D172) A polynucleotide encoding a transmembrane domain CD20t11); and o r SEQ ID NO: 130 (CD20t(C167~C183), SG4S linker (SEQ ID NO: 2 23), and CD8α(P120~V201) A polynucleotide encoding a transmembrane domain CD20t 12) is encoded by a polynucleotide comprising a nucleotide sequence selected from the list consisting of In certain embodiments, the polypeptide construct comprising CD20t is SEQ ID NO: 11 3 (CD20t3; CD20t(K142~S188) and CD8α(I183~T2 03) transmembrane domain); SEQ ID NO: 121 (CD20t(P160~Q187), SG4 S linker (SEQ ID NO: 223), and the transmembrane domain of CD28 (I96 - D172) encoding CD20t7); SEQ ID NO: 123 (CD20t concatamer (P160 - Q187) separated by SGS linker , SG4S linker (SEQ ID NO: 223), and encoding the transmembrane domain of CD28 (I96 - D172) CD20t8); SEQ ID NO: 1 25 (CD20t (P160 - Q187), SG4S linker (SEQ ID NO: 223), and encoding the transmembrane domain of CD8α (P120 - V201) CD20t9); SEQ ID NO : 127 (CD20t (C167 - C183), SG4S linker (SEQ ID NO: 223), and encoding the transmembrane domain of CD28 (I96 - D172) CD20t10); SEQ ID NO: 129 (CD20 concatamer (C167 - C183) separated by SG4S linker (SEQ ID NO: 223) , SG4S linker (SEQ ID NO: 223), and CD28( I96 - D172) transmembrane domain encoding CD20t11); and SEQ ID NO: 13 1 (CD20t (C167 - C183), SG4S linker (SEQ ID NO: 223), and encoding the transmembrane domain of CD8α (P120 - V201) CD20t12)) consisting of a list selected from the amino acid sequence containing.

[0159] The polypeptide construct comprising the CD20t polypeptide may comprise any signal peptide capable of directing the polypeptide construct to the cell surface. For example, a polypeptide construct comprising a CD20 t polypeptide (e.g., a polypeptide containing an amino acid sequence selected from the list consisting of SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220)) is SEQ ID NO: 2 (GMCSFRα), SEQ ID NO: 4 (IG kappa), SEQ ID NO: 6 (immunoglobulin ), SEQ ID NO: 38 (GMCSFRα), SEQ ID NO: 4 (IG kappa), SEQ ID NO: 6 (immunoglobulin (B lymphocyte antigen CD20t), SEQ ID NO: 8 (CD8α), SEQ ID NO: 10 (TVB2), SEQ ID NO: 12 (CD 52), or an amino acid sequence selected from the list consisting of SEQ ID NO: 14 (LNGFR), with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or also 100% identical amino acid sequence can be fused with a signal peptide containing the same. It is possible.

[0160] The polypeptide construct containing the CD20t polypeptide may contain a dimerization domain or optionally may contain any transmembrane domain, including a transmembrane domain that does not contain a dimerization domain. For example, a polypeptide construct containing the CD20t polypeptide (e.g., an amino acid sequence selected from the list consisting of SEQ ID NO: 21 8, SEQ ID NO: 219, and SEQ ID NO: 220) of the polypeptide), SEQ ID NO: 26 (glycophorin A E91~R116), SEQ ID number 28 (glycophorin A I92~I114), SEQ ID NO: 30 (glycophorin A (I9 2~L109), integrin β3 (A737~W741), SEQ ID NO: 32 (CD3 zeta chain), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTL A4) and SEQ ID NO: 40 (LNGFR), with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical amino acid sequence can be fused with a transmembrane domain containing the same. The polypeptide construct containing CD20t may further contain a transmembrane domain derived from CD28 and / or CD8α or homologous thereto. It is possible.

[0161] ​​A polypeptide construct comprising a CD20t polypeptide can include any peptide linker (or, in some embodiments, may not include a peptide linker). For example, CD2 A polypeptide construct comprising a 0t polypeptide (e.g., a polypeptide comprising an amino acid sequence selected from the list consisting of SEQ ID NO: 218, SEQ ID NO: 219 , and SEQ ID NO: 220) is at least 70%, 75%, 80%, 85% Identity with an amino acid sequence selected from the list consisting of SEQ ID NO: 16 (GSG), SEQ ID NO: 18 (SGSG), SEQ ID NO: 20 ((G4S) 3), SEQ ID NO: 22 ((G4S)4) and SEQ ID NO: 24 (Whitlow) A peptide comprising an amino acid sequence having 90%, 95%, 99%, or 100% Can be fused to a peptide linker.

[0162] Another example of a CD polypeptide that can be truncated and incorporated into a cell tag described herein Is CD52. CD52 is, at its C-terminus, a 12-amino acid peptide linked to a glycosylphosphatidyl Inositol (GPI) anchor, Occurs endogenously in humans. In some embodiments, glycosylphosphatidylinositol Tol (GPI) can be used to anchor the polypeptides described herein to the cell surface Can be called.

[0163] As used herein, a polypeptide construct comprising a CD52 polypeptide in which any amino acid, domain, or fragment of endogenous CD52 has been truncated Is presented. As used herein , The CD52 polypeptide can include a truncated CD52 (CD52t) polypeptide In some embodiments, the CD52 polypeptide comprises a cell comprising the CD52t polypeptide​ It consists of or consists essentially of cell surface polypeptides. In other embodiments, C The D52 polypeptide may include cell surface polypeptides in addition to other CD52 domains (e.g., the CD52 signal peptide), including the CD52t polypeptide.

[0164] Disclosed herein are polypeptide constructs comprising cell surface polypeptides, including truncated CD52t polypeptides. In some embodiments, a CD52 signal peptide is linked to the CD52t polypeptide to direct the truncated variant to the cell surface. In some embodiments, the cell surface polypeptide comprising the CD52t polypeptide may incorporate one or more epitopes that can be recognized by an exogenously introduced antibody or binding partner. For example, the CD52t polypeptide may incorporate one or more alemtuzumab-binding domains, thereby facilitating targeted depletion of cells expressing the polypeptide constructs described herein. In some embodiments, the targeted depletion may result from CDC and / or ADCC mediated by alemtuzumab, or from another cellular mechanism that mediates the cytotoxic effect of recognition by alemtuzumab. Non-limiting examples of anti-CD52 molecules that can recognize cell surface polypeptides comprising the CD52t polypeptide include alemtuzumab, ANT1034, HI186 (Bio Rad), YTH34.5 (Bio Rad), and YTH66.9HL ( Bio-Rad). In various embodiments, the antibody can be a monoclonal antibody, scFv, scFab, diabody, or camelid antibody. In certain embodiments , The polynucleotide sequence encoding the CD52 epitope has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identity to the nucleotide sequence of SEQ ID NO: 142, or 100% and includes a nucleotide sequence. In certain embodiments, the CD 52 epitope has at least 70%, 75% identity to the amino acid sequence of SEQ ID NO: 143, 80%, 85%, 90%, 95%, 99% , or 100%.

[0165] The polypeptide constructs described herein can have multiple epitopes that can be recognized by an antibody or binding partner. For example, in the case of the CD52t polypeptide, a polypeptide construct containing the CD5 2t polypeptide can contain multiple epitopes specific to a binding partner (e.g., alemtuzumab ). In certain embodiments, the CD52t polypeptide can contain multiple copies (e.g., at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, or at least 10 copies) of the amino acid sequence shown in SEQ ID NO: 143. Each copy or repeat of the amino acid sequence can be separated by a linker (e.g., a Whitlow linker) within the polypeptide construct. In certain embodiments, the polypeptide construct is linked to a transmembrane domain (e.g., the CD28 transmembrane domain or a fragment thereof) (e.g., via one or more copies of the (G4S) linker (SEQ ID NO: 221)) and fused to one or more copies of the CD52t polypeptide linked to the CD52 signal peptide. In certain embodiments, a polypeptide construct containing the CD52t polypeptide contains a CD52 signal peptide that is linked to one or more copies of the CD52t polypeptide fused to a Whitlow linker and linked to a transmembrane domain (e.g., the CD28 transmembrane domain or a fragment thereof). In certain embodiments, a polypeptide construct containing the CD52t polypeptide SEQ ID NO: 144 (CD52t1; encoding 1 copy of the epitope / linker), SEQ ID NO: 146 (CD52t2; encoding 2 copies of the epitope / linker), and SEQ ID NO: 148 (CD52t3; encoding 3 copies of the epitope / linker), and is encoded by a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of. In certain embodiments, the polypeptide comprising the CD52t polypeptide is SEQ ID NO: 145 (CD52t1; encoding 1 copy of the epitope / linker), SEQ ID NO: 147 (CD52t2; encoding 2 copies of the epitope / linker), and SEQ ID NO: 149 (CD52t3; encoding 3 copies of the epitope / linker), and has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%

[0166] identity to an amino acid sequence selected from the list consisting of. The polypeptide construct comprising the CD52t polypeptide can include any transmembrane domain, including a transmembrane domain that includes a dimerization domain or does not include a dimerization domain. For example, a cell surface polypeptide comprising the CD52t polypeptide can be fused via a linker (e.g., a 3×GS linker (SEQ ID NO: 224)) to a transmembrane t4; CD52 signal peptide, 3×GS peptide linker (SEQ ID NO: 224), and glycophorin A transmembrane domain), SEQ ID NO: 151 (CD52t5; CD52 signal peptide, 3×GS peptide linker (SEQ ID NO: 224), and glycophorin A transmembrane domain), and SEQ ID NO: 152 (CD52t6; CD52 signal peptide, 3×G S linker (SEQ ID NO: 224), and glycophorin A-integrin β3 transmembrane domai n) is selected from the list consisting of amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity.

[0167] In multiple embodiments, a polypeptide construct comprising a CD52t polypeptide (e.g., an amino acid sequence selected from the list consisting of SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149) having at least 70%, 75%, 80%, 85%, 90%, 95%, 9 9%, or 100% identity, the sequence number 143 that can be linked to the CD52 signal peptide is SEQ ID NO: 26 (glycophorin A E91 ~R116), SEQ ID NO: 28 (glycophorin A I92~I114), SEQ ID NO: 30 (g lycophorin A (I92~L109). integrin β3 (A737~W741), SEQ ID num ber 32 (CD3 zeta chain), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTLA4) and SEQ ID NO: 40 (LNGFR) is selected from the list consisting of amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100% identity and can be linked to a transmembrane domain comprising an amino acid sequence. ​

[0168] A polypeptide construct comprising a CD52t polypeptide can include any signal peptide capable of directing the polypeptide construct to the cell surface. In certain embodiments , the CD52t polypeptide is linked to the signal peptide of CD52 (e.g., the polypeptide construct comprises an amino acid sequence having at least 70%, 75%, 80%, 85% , 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149). In some embodiments, the polypeptide construct comprises a CD52t polypeptide linked to a signal peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GMCSFRα), SEQ ID NO: 4 (IG kappa), SEQ ID NO: 6 (immunoglobulin E), SEQ ID NO: 8 (CD8α), SEQ ID NO: 10 (TVB2), SEQ ID NO: 12 (CD52), or SEQ ID NO: 14 (LNFGR) (e.g., SEQ ID NO: 143). (IG kappa), SEQ ID NO: 6 (immunoglobulin E), SEQ ID NO: 8 (CD8α), SEQ ID NO: 10 (TVB2), SEQ ID NO: 12 (CD52), or SEQ ID NO: 14 (LNFGR) and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GMCSFRα), SEQ ID NO: 4 (IG kappa), SEQ ID NO: 6 (immunoglobulin E), SEQ ID NO: 8 (CD8α), SEQ ID NO: 10 (TVB2), SEQ ID NO: 12 (CD52), or SEQ ID NO: 14 (LNFGR).

[0169] A polypeptide construct comprising a CD52t polypeptide can include any peptide linker (or, in some embodiments, may not include a peptide linker). For example, a cell surface polypeptide comprising CD5 2t (e.g., comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 143) is linked to SEQ ID NO: 16 (GSG), SEQ ID NO: 18 (SG ), at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 16 (GSG), SEQ ID NO: 18 (SG SG), SEQ ID NO: 20 ((G4S)3), SEQ ID NO: 22 ((G4S)4) and SEQ ID NO: selected from the list consisting of 24 (Whitlow), at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity and can be fused with a peptide linker containing an amino acid sequence.

[0170] In other embodiments, the cell surface polypeptide is a tumor necrosis factor (TNF) receptor super family may include a truncated form of the polypeptide derived from. For example, LNGFR is , due in part to the formation of disulfide bonds between the cysteine residues of the protein, a single-pass transmembrane type I transmembrane glycoprotein having an extracellular domain that exhibits a folded structure. In certain embodiments, the polynucleotide encoding LNGFR or a portion thereof is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the list consisting of SEQ ID NO: 153, SEQ ID NO: 155 (encoding K29-N250 of the LNGFR extracellular domain), SEQ ID NO: 157 (encoding E65-N250 containing cysteine residues 2, 3, 4 capable of forming disulfide bonds), and SEQ ID NO: 159 (encoding R108-N250 containing cysteine residues 3, 4 capable of forming disulfide bonds). has a nucleotide sequence. In certain embodiments, LNGFR is SEQ ID NO: 154, SEQ ID NO: 156 (including K29-N250 of the LNGFR extracellular domain), SEQ ID NO: 158 (E65- including cysteine residues 2, 3, 4 capable of forming disulfide bonds 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity and includes a nucleotide sequence. In certain embodiments, LNGFR is SEQ ID NO: 154, SEQ ID NO: 156 (including K29-N250 of the LNGFR extracellular domain), SEQ ID NO: 158 (E65- including cysteine residues 2, 3, 4 capable of forming disulfide bonds (including N250), and selected from the list consisting of R108 to N250 (including cysteine residues 3 and 4 capable of forming disulfide bonds). At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence.

[0171] As used herein, a polypeptide construct comprising a LNGFR polypeptide in which any amino acid, domain, or fragment of endogenous LNGFR is truncated is presented. As used herein, the LNGFR polypeptide may include the LNGFRt polypeptide. In some embodiments, the LNGFR polypeptide consists of or consists essentially of a cell surface polypeptide comprising the LNGFRt polypeptide. In other embodiments, the LNGFR polypeptide may include a cell surface polypeptide comprising the LNGFRt polypeptide in addition to other LNGFR domains (e.g., the LNGFR transmembrane domain).

[0172] The polypeptide construct may include a truncated LNGFR (LNGFRt herein) polypeptide in which any domain or fragment thereof is truncated compared to the wild-type protein. For example, the LNGFRt polypeptide may truncate one or more of the transmembrane domain or a portion thereof, the intracellular domain or a portion thereof, or the extracellular domain or a portion thereof. In some embodiments, the LNGFRt polypeptide truncates one or more of the TNFR-Cys repeats that form the native binding domain for its ligand (e.g., NGF, BDNF, NTF3, and NTF4). ​​​​​​​​In one embodiment, the polypeptide construct can be composed of the entire extracellular domain ( In one embodiment, the LNGFRt polypeptide comprises the LNGFRt polypeptide comprising SEQ ID NO: 156. Polypeptide constructs containing Rt polypeptides are fused to the LNGFR transmembrane domain In one embodiment, the LNGFRt polypeptide is linked to the LNGFR transmembrane domain. The polynucleotide encoding the LNGFR t1) at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or a nucleotide sequence having 100% identity. The LNGFRt polypeptide is represented by the sequence of SEQ ID NO: 162, linked to the FR transmembrane domain. At least 70%, 75%, 80%, 85%, It has 90%, 95%, 99% or 100% identity.

[0173] The polypeptide construct comprising the LNGFRt polypeptide may comprise a dimerization domain or or any transmembrane domain, including a transmembrane domain that does not include a dimerization domain. For example, the polypeptide construct can be linked to the LNGFR transmembrane domain or a fragment thereof. In another embodiment, the LNGFRt polypeptide may include a ligated LNGFRt polypeptide. The peptide is then coupled to a transmembrane domain derived from a different polypeptide, the transmembrane domain being capable of dimerization. The polypeptide can be fused (e.g., via a linker) to a signaling domain or fragment thereof. In one embodiment, the polypeptide construct comprising the LNGFRt polypeptide is SEQ ID NO: 16. 3 (LNGFRt2; the entire LNGFR extracellular domain; GS linker and CD28 transmembrane domain) (Domain); SEQ ID NO: 164 (LNGFRt3; a fragment of the LNGFR extracellular domain containing cysteine residues 2 - 4, GS peptide linker, and CD28 transmembrane domain), SEQ ID NO: 165 (LNGFRt3; a fragment of the LNGFR extracellular domain containing cysteine residues 3 - 4, GS peptide linker, and CD28 transmembrane domain); SEQ ID NO: 166 (LNGFRt5; a fragment of the LNGFR extracellular domain containing cysteine residues 3 - 4, GS linker, and glycophorin A transmembrane domain), SEQ ID NO: 167 (LNGFRt6; a fragment of the LNGFR extracellular domain containing cysteine residues 3 - 4, GS linker, and glycophorin A transmembrane domain); and SEQ ID NO: 168 (LNGFRt7; a fragment of the LNGFR extracellular domain containing cysteine residues 3 - 4, GS linker, and glycophorin A - integrin β3 transmembrane domain), having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 164 - 168. In some embodiments, a polypeptide construct comprising an LNGFRt polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 26 (glycophorin A E91 - R116), SEQ ID NO: 28 (glycophorin A I92 - I114), SEQ ID NO: 30 (glycophorin A (I92 - L109). integrin β3 (A737 - W741), SEQ ID NO: 32 (CD3 zeta chain), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTLA4), and SEQ ID NO: 40 (LNGFR).

[0174] In some embodiments, a polypeptide construct comprising an LNGFRt polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 26 (glycophorin A E91 - R116), SEQ ID NO: 28 (glycophorin A I92 - I114), SEQ ID NO: 30 (glycophorin A (I92 - L109). integrin β3 (A737 - W741), SEQ ID NO: 32 (CD3 zeta chain), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTLA4), and SEQ ID NO: 40 (LNGFR). In some embodiments, a polypeptide construct comprising an LNGFRt polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 26 (glycophorin A E91 - R116), SEQ ID NO: 28 (glycophorin A I92 It can be fused with a transmembrane domain containing an acid sequence.

[0175] A polypeptide construct comprising an LNGFRt polypeptide can include any signal peptide capable of directing the polypeptide construct to the cell surface. For example, an LNG FRt polypeptide-containing cell surface polypeptide can be fused to an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GMCSFRα), SEQ ID NO: 4 (IG kappa), SEQ ID NO: 6 (immunoglobulin E), SEQ ID NO: 8 (CD8α), SEQ ID NO: 10 (TVB2), SEQ ID NO: 12 (CD52), or SEQ ID NO: 14 (LNFG R). A polypeptide construct comprising an LNGFRt polypeptide can include any peptide linker (or, in some embodiments, may not include a peptide linker). For example, an LN

[0176] GFRt polypeptide-containing cell surface polypeptide can be fused to an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 10 0% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 16 (GSG), SEQ ID NO: 18 (SGSG), SEQ ID NO: 20 ((G4S)3), SEQ ID NO: 22 ((G4S)4), and SEQ ID NO: 24 (Whitlow). A polypeptide construct comprising an LNGFRt polypeptide can be fused to a peptide linker comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 10 0% identity to an amino acid sequence selected from the list consisting of SEQ ID NO: 16 (GSG), SEQ ID NO: 18 (SGSG), SEQ ID NO: 20 ((G4S)3), SEQ ID NO: 22 ((G4S)4), and SEQ ID NO: 24 (Whitlow).

[0177] Polynucleotide construct Vector The polypeptide constructs described herein can be incorporated into engineered cells via a vector. ​may be encoded by one or more polynucleotides. As used herein, "expression vector" or "vector" refers to any genetic element that behaves as an autonomous unit of polynucleotide replication within a cell (i.e., is capable of replicating under its own control) or is capable of replication by insertion into the chromosome of a host cell, such as a plasmid, chromosome, virus, transposon, to which another polynucleotide segment is joined so as to effect replication and / or expression of the joined segments. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and cosmids. A vector may contain polynucleotide sequences necessary to effect ligation or insertion of the vector into a desired host cell and to effect expression of the joined segments. Such sequences will vary depending on the host organism and include promoter sequences to effect transcription, enhancer sequences to increase transcription, sequences for ribosome binding sites, and transcription / translation termination sequences. Alternatively, an expression vector may be capable of directly expressing the

[0178] product of the nucleic acid sequence encoded therein without ligation or integration of the vector into the DNA sequence of the host cell. A vector may also include a "selectable marker gene." As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that enables a Publication No. 1992 / 08796 Pamphlet and Publication No. 1994 / 28143 Pamphlet , Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980), O’Hare et al. , Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mulligan & Berg, Proc. Natl. Acad . Sci. USA, 78: 2072 (1981), Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981 ), Santerre et al., Gene, 30: 147 (1984), Kent et al., Science, 237: 901-903 (19 87), Wigler et al., Cell, 11: 223 (1977), Szybalska & Szybalski, Proc. Natl. Aca d. Sci. USA, 48: 2026 (1962), Lowy et al., Cell, 22: 817 (1980), and U.S. Pat ent Nos. 5,122,464 and 5,770,359 are described therein.

[0179] In some embodiments, the vector is capable of replicating within a host cell and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selection pressure, an "episomal expression vector" or "episome" (see, e.g., Conese et al., Gene Therap y, 11:1735-1742 (2004)). Representative, commercially available episomal expression vectors are , Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EB V) V) episomal plasmids using the origin of replication (oriP), including but not limited to . Vectors such as pREP4, pCEP4, pREP7, and pcDNA3.1 manufactured by Invitroge n (Carlsbad, Calif.), and pBK-CMV manufactured by Stratagen e (La Jolla, Calif.) represent non-limiting examples of episomal vectors that use the T antigen and the origin of replication of SV40 instead of EBNA1 and oriP.

[0180] As used herein, a polypeptide construct can include a signal peptide, a transmembrane domain, a truncated form mutant of a native polypeptide as a cell surface polypeptide, and optionally a peptide linker that connects the transmembrane domain to the cell surface polypeptide. For example, FIG. 4A illustrates an embodiment of a polypeptide construct having the functionality of a cell tag. The truncated form mutant extends to the extracellular matrix via a peptide linker that is connected to the transmembrane domain. The truncated form mutant can include one or more epitopes for an anti-truncated form mutant antibody (e.g., exogenously added) that can recognize and bind to the truncated form mutant / cell tag. In some embodiments, the binding of the antibody to the truncated form mutant results in depletion of the cells (e.g., via ADCC), which can be beneficial during adoptive cell therapy, such as operating the cells to express one or more additional molecules such as cytokines, TCRs, and / or CARs. In some embodiments, different polypeptide constructs include certain transmembrane domains and signal peptides, but the truncated forms incorporated into the polypeptide construct forms and signal peptides, but the truncated Vary the identification of the T-shaped variants. For example, different polypeptide constructs may contain different truncated variants of the same native polypeptide. When two polypeptide constructs incorporate different truncated variants of the same native polypeptide, the polypeptide constructs may further differ based on the presence / absence or length of the peptide linker within the polypeptide construct. In some embodiments, the peptide linker of each polypeptide construct is sized to maintain a relatively constant distance between the distal end of a particular truncated variant and the cell surface.

[0181] As used herein, polynucleotides and methods are provided for facilitating the construction of polypeptide constructs for use during immunotherapy. In certain embodiments, the polynucleotide can include a vector that includes a sequence encoding a signal peptide and a transmembrane domain (e.g., including or lacking a transmembrane dimerization domain). The vector can result in the cloning of an insert between the signal peptide and the transmembrane domain such that the insert includes a nucleotide sequence encoding a particular truncated variant and optionally a peptide linker. Alternatively, a vector can be provided that includes a polynucleotide encoding a signal peptide, a particular truncated variant, and optionally a linker, and that results in the insertion of a nucleotide sequence encoding a transmembrane domain adjacent to the coding sequence of the truncated variant or linker. One or more particular domains within the resulting polypeptide construct are maintained consistently, but domains that can be variable between polypeptide constructs are presented. ​ By presenting a series of vectors that enable insertion, the present disclosure provides any combination of signal peptides, truncated mutants, transmembrane domains, and linkers, facilitating the production of polypeptide constructs. Including any combination of signal peptides, truncated mutants, transmembrane domains, and linkers, facilitating the production of polypeptide constructs. Making it easier to produce polypeptide constructs.

[0182] Modification of Vectors Polynucleotide vectors useful in the methods and compositions described herein can be vectors that comply with the "Standards for the Manufacture and Quality Control of Pharmaceuticals and Pharmaceutical Excipients" (GMP). For example, GMP vectors can be purer than non-GMP vectors. In some cases, purity can be measured by bioburden. For example, bioburden can be the presence or absence of aerobic bacteria, anaerobic bacteria, spore-forming bacteria, fungi, or combinations thereof in the vector composition. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, purity can be measured by bioburden. For example, bioburden can be the presence or absence of aerobic bacteria, anaerobic bacteria, spore-forming bacteria, fungi, or combinations thereof in the vector composition. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, purity can be measured by bioburden. For example, bioburden can be the presence or absence of aerobic bacteria, anaerobic bacteria, spore-forming bacteria, fungi, or combinations thereof in the vector composition. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. In some cases, pure vectors can be low endotoxin or endotoxin-free. Purity can also be measured by double-stranded primer walking sequencing. The identity of the plasmid can be a source for determining the purity of the vector. The GMP vectors of the present invention can be 10% - 99% purer than non-GMP vectors. GMP vectors can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purer than non-GMP vectors, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof.

[0183] In some cases, a terminator sequence at the end of the first gene program is used. 。The terminator sequence can ensure that the transcript terminates before the second gene program starts. For example, an expression vector may contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are available from the 5' untranslated region of eukaryotic or viral DNA or cDNA, and sometimes also from the 3' untranslated region. These regions may contain nucleotide segments that are transcribed as polyadenylated fragments within the untranslated portion of the mRNA. Cells containing the expression vector are grown under conditions, in vivo or in vitro, that result in the expression of the desired polypeptide.

[0184] In some cases, a spacer sequence can be used at the end of the first polypeptide encoded by a polynucleotide in the vector. In other cases, a spacer sequence can be used at the end of the second gene in the vector. A spacer sequence can also be used subsequent to the first and second genes in the vector.

[0185] Using these vectors, a gene of interest, or a polypeptide encoded by a portion of the gene of interest, can be expressed. The gene or portion of the gene can be inserted using any method, viral or non-viral. For example, the method can be a non-viral-based technique.

[0186] Additional features encoded by the construct The polynucleotides disclosed herein can encode one or more proteins in addition to, or together with, the polynucleotide constructs described above. For example, some In embodiments, the polypeptide construct is a chimeric antigen receptor (CAR), a T cell receptor (T CR), and / or cytokines.

[0187] Chimeric Receptors Some embodiments described herein include a chimeric receptor encoding a chimeric receptor expressed on the cell surface. In some cases, the chimeric receptor comprises a polynucleotide that binds an antigen, e.g., a tumor Antibodies capable of recognizing and binding to tumor antigens, such as associated antigens or tumor-specific antigens. In some cases, the antigen-binding region may be an antibody or binding fragment, e.g. For example, Fab, Fab', F(ab')2, F(ab')3, scFv, sc(Fv)2, dsFv, diabodies, minibodies, and nanobodies or binding fragments thereof Optionally, the antigen-binding region comprises an scFv. Optionally, the chimeric receptor The receptor comprises an scFv (e.g., a chimeric antigen receptor (CAR)). Chimeric antigen receptors include pattern recognition receptors. In other cases, chimeric receptors are engineered T Contains the T cell receptor (TCR).

[0188] Chimeric antigen receptors (CARs) In some embodiments, the cells expressing the polypeptide constructs described herein also , they also express one or more chimeric antigen receptors (CARs).

[0189] The chimeric antigen receptors (CARs) described herein are capable of delivering exogenous specificity to immune effectors. CARs are engineered receptors that are grafted onto target cells. In some cases, CARs are antigen-binding The functional domain, stalk region, transmembrane domain, and intracellular domain (endodomain) comprises an extracellular domain (ectodomain). In some cases, the intracellular domain further comprises one or more intracellular signaling domains. In some cases, the CARs described herein comprise an antigen-binding domain, a stalk region, a transmembrane domain, one or more co-stimulatory domains, and a signaling domain for T cell activation.

[0190] In multiple embodiments, the CARs of the present disclosure otherwise comprise a target-specific binding element, otherwise referred to as an antigen-binding moiety. In multiple embodiments, the CARs of the present disclosure are engineered to target a desired tumor antigen by engineering a desired antigen-binding moiety that specifically binds to a given antigen on tumor cells. In the context of the present disclosure, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to specific hyperproliferative disorders such as cancer. The antigen-binding domain can include complementarity-determining regions of monoclonal antibodies, variable regions of monoclonal antibodies, and / or antigen-binding fragments thereof. Complementarity-determining regions (CDRs)

[0191] are short amino acid sequences found within the variable domains of antigen receptor (e.g., immunoglobulin and T cell receptor) proteins that are complementary to the structure of an antigen and thus present a structure to the receptor that confers its specificity for this particular antigen. Each polypeptide chain of an antigen receptor can contain three CDRs (CDR1, CDR2, and CDR3). In some cases, the antigen-binding domain includes F(ab’)2, Fab’, Fab, Fv, or scFv. Optionally, the antigen-binding domain is an amino acid sequence that presents a structure to the receptor that confers its specificity for this particular antigen. Each polypeptide chain of an antigen receptor can contain three CDRs (CDR1, CDR2, and CDR3). In some cases, the antigen-binding domain includes F(ab’)2, Fab’, Fab, Fv, or scFv. In some cases, the antigen-binding domain comprises F(ab’)2, Fab’, Fab, Fv, or scFv. Optionally, the antigen-binding domain , it is an scFv. In some cases, the antigen-binding domain is a Fab. In some cases , the antigen-binding domain is a Fab'. In some cases, the antigen-binding domain is F( ab')2. In some cases, the antigen-binding domain is an Fv.

[0192] In some embodiments, the CARs described herein are CD19, CD20, CD33 , CD44, BCMA, CD123, EGFRvIII, α-folate receptor, CAIX, C D30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin , CD22, EGFR, MUC-1, MUC-16, MAGE-A1, h5T4, PSM A, TAG-72, or an antigen-binding domain that binds to an epitope in VEGF-R2 . In some embodiments, the CARs described herein are CD19, CD33 , BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EG P-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC- 1, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvI II, CD123, and an antigen-binding domain that binds to an epitope on VEGF-R2 . In some embodiments, the CARs described herein include an antigen-binding domain that binds to an epitope on CD19 or CD33 . In some cases, the CARs described herein include an antigen-binding domain that binds to an epitope on CD19 . In some cases, the CARs described herein include an antigen-binding domain that binds to an epitope on CD19 Therefore, the CAR described herein has an antigen-binding domain that binds to an epitope on CD33. In further embodiments, the CAR or chimeric receptor or antigen-binding polypeptide described herein comprises an autoantigen-binding region or antigen-binding region that binds to an epitope on HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, or type II collagen.

[0193] In some embodiments, the polynucleotides, polypeptides, and methods described herein can be used for the treatment of proliferative diseases such as cancer, the treatment of autoimmune diseases, or the treatment of infections such as viral infections, bacterial infections, or parasitic infections. In some aspects, the antigen is an antigen that is increased in cancer cells, autoimmune cells, or cells infected with a virus, bacterium, or parasite. Pathogens that can be targeted include, without limitation, the genus Plasmodium, trypanosomes, the genus Aspergillus, the genus Candida, hepatitis A virus, hepatitis B virus, hepatitis C virus, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or the Ebola pathogen. Autoimmune diseases can include graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, Crohn's disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, or autoimmune uveitis.

[0194] The pathogen essentially recognized by the CAR can be any type of pathogen, but in some embodiments, the pathogen is a fungus, bacterium, or virus. Exemplary viral pathogens include those of the family Adenoviridae, Epstein - Barr virus (EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, BK virus, HPV, HSV, viruses of the HHV family, viruses of the hepatitis family, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, Ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, and... Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, and... Pseudomonas), Shigella, Campylobacter, Staphylococcus, Helicobacter, Escherichia coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella. In some embodiments, the pathogen receptor dectin 1 is used to create a CAR that recognizes carbohydrate structures on the cell wall of fungi such as Aspergillus. In another embodiment, a CAR that blocks viral infection and viral pathogenesis can be made based on an antibody that recognizes viral determinants (e.g., glycoproteins derived from CMV and Ebola). In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin.

[0195] In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. In some embodiments, a "stalk" region or "spacer" region or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" includes any oligonucleotide or polypeptide that functions in the polypeptide chain to link the transmembrane domain to the extracellular domain or the cytoplasmic domain. In some embodiments, the stalk domain is flexible enough to allow the antigen-binding domain to face in different directions to facilitate antigen recognition. In some cases, the stalk region includes a hinge region derived from IgG1. Alternatively, the stalk region is the CH2CH3 region of an immunoglobulin. and, optionally, a portion of CD3. Optionally, the stalk region is the CD8α hinge region, I described in International Publication No. 2 the 12 - amino - acid hinge region of IgG4 - Fc (ESKYGPPCPPCP (SEQ ID NO: 225 )), or an IgG4 hinge region, as described in International Publication No. 2016 / 073755 pamphlet.

[0196] The transmembrane domain may be derived from a natural source or a recombinant source. When the source is natural, the domain may be derived from any membrane - binding protein or transmembrane protein. Suitable transmembrane domains may include the transmembrane regions of the alpha, beta, or zeta chains of the T - cell receptor, and may also include the transmembrane regions derived from CD28, CD3 epsilon, CD 3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In some embodiments, at one or both ends of the synthetic transmembrane domain, triplets of phenylalanine, tryptophan, and valine are found. Optionally, in some embodiments, a short oligonucleotide linker or polypeptide linker, which is between 2 and 10 amino acids in length, can form a linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In some embodiments, the linker is a glycine - serine linker. In some embodiments, the transmembrane domain includes a CD8α transmembrane domain or a CD3ζ transmembrane domain. In some embodiments, the transmembrane domain includes a CD8α transmembrane domain. In other embodiments ​ The transmembrane domain includes the CD3ζ transmembrane domain. In yet other embodiments, the transmembrane domain includes a transmembrane dimerization domain.

[0197] The intracellular domain may include one or more co-stimulatory domains. Exemplary co-stimulatory domains include CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10 , DAP12, OX40 (CD134), or fragments or combinations thereof, but are not limited thereto. Optionally, the CARs described herein include one or more, or two or more, of the co-stimulatory domains selected from CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof. Optionally, the CARs described herein include one or more, or two or more, of the co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS . Optionally, the CARs described herein include one or more, or two or more, of the co-stimulatory domains selected from CD8, CD28, 4-1BB (CD137), or fragments or combinations thereof. Optionally, the CARs described herein include one or more, or two or more, of the co-stimulatory domains selected from CD28, 4-1BB (CD137), or fragments or combinations thereof. Optionally, the CARs described herein include one or more, or two or more, of the co-stimulatory domains selected from the co-stimulatory domain CD that is CD28, 4-1BB (CD137), or fragments or combinations thereof. Optionally, the CARs described herein are co-stimulatory domains and are CD ​It comprises 28 and 4-1BB (CD137), or respective fragments thereof. Optionally, the CARs described herein comprise the co-stimulatory domains CD28 and OX40 ( CD134), or respective fragments thereof. Optionally, the CARs described herein comprise the co-stimulatory domains CD8 and CD28, or respective fragments thereof. Optionally, the CARs described herein comprise the co-stimulatory domain CD 28, or a fragment thereof. Optionally, the CARs described herein comprise the co-stimulatory domain 4-1BB (CD137), or a fragment thereof. Optionally, the CARs described herein comprise the co-stimulatory domain OX40 (CD134), or a fragment thereof. Optionally, the CARs described herein comprise the co-stimulatory domain CD8 , or a fragment thereof.

[0198] Intracellular signaling domains, also known as cytoplasmic domains of the CARs of the present disclosure, contribute to the activation of at least one of the normal effector functions of immune cells containing the CAR. The term "effector function" refers to the specialized functions of cells. The effector functions of T cells can be, for example, cytotoxic activity or helper activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits effector function signals and directs the cell to perform specialized functions. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used to transmit effector function signals, such truncated... The caged portion can be used in place of the intact chain. Thus, the intracellular sig The term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transmit an effector function signal. In some embodiments, the intracellular domain further includes a signaling domain for T cell activation. Optionally, the signaling domain for T cell activation includes a domain derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, C D5, CD22, CD79a, CD79b, or CD66d. Optionally, the signaling domain for T cell activation includes a domain derived from CD3 zeta.

[0199] The term "functional portion," as used with respect to a CAR, refers to any portion or fragment of the CAR of the present disclosure that retains the biological activity of the CAR (parent CAR) of which the functional portion is a part. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional portion of the CAR can encode a polypeptide that includes, for example, about 10%, 25% , 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0200] As used herein, the term "functional variant" refers to a polypeptide or protein that has substantial or significant sequence identity or sequence similarity to a reference polypeptide and retains the biological activity of the reference polypeptide of which it is a variant. A functional variant is, for example, a variant of a CAR (parent CAR) described herein that binds to a target cell ​ include variants that retain the ability to recognize at the same level as, to the same extent as, or to a higher extent than the parental CAR. With reference to the nucleic acid sequence encoding the parental CAR, the nucleic acid sequence encoding a functional variant of the CAR may be, for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parental CAR. The polynucleotide constructs disclosed herein can be co-expressed with the CAR in engineered cells. In some embodiments, the polypeptide construct and the CAR can be encoded by a single transcript. The advantage of encoding a polypeptide construct that contains a truncated variant and the CAR within the same transcript is that the engineered cell that produces the CAR protein is also likely to produce the polypeptide construct. Thus, in the event that an intervention is needed to reduce the expression of the CAR (e.g., to mitigate the side effects of treatment) during immunotherapy, the engineered cells co-expressing the polypeptide construct and the CAR can be primed to respond within a relatively short time frame by administration of an exogenous antibody that targets the cell tag conferred by the truncated variant disclosed herein.

[0201]

[0202] CD19-specific CAR CD19 is a cell surface glycoprotein of the immunoglobulin superfamily. In some cases, CD19 has been detected within solid tumors such as pancreatic cancer, liver cancer, and prostate cancer.

[0203] In some embodiments, the antigen-binding portion of the CAR described herein is specific for CD19.​​​​​​​​​​​​​​ is different. When expressed on the cell surface, the CD19-specific CAR redirects the specificity of T cells to human CD19. In multiple embodiments, the antigen-binding domain is a variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen-specific, anti-CD19 monoclonal antibody, connected by a glycine-serine linker or a whitlow linker any flexible linker, and includes a single-chain antibody fragment (scFv) containing VL and VH. In multiple embodiments, the scFv is SJ25C1 and / or FMC63. In multiple embodiments, the scFv is a humanized scFv. In some embodiments, the antigen binding moiety can include VH and VL connected directionally, for example, from the N-terminus to the C-terminus, as VH-linker-VL or VL-linker-VH.

[0204] In some embodiments, described herein is a CD19-specific CAR in which the antigen-binding domain includes a scFv that binds CD19. In some cases, the antigen-binding domain recognizes an epitope on CD19.

[0205] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some embodiments, described herein is a CD19-specific CAR-T cell in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). recognized. It can be. In some cases, the CD19-specific CAR-T cells have a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134 ), or one or more co-stimulatory domains selected from fragments or combinations thereof ; and further include a signaling domain derived from CD3 zeta.

[0206] In some embodiments, the CD19-specific CAR-T cells described herein include the scF v antigen-binding domain, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR01 5, JCAR017, or the 19-28z CAR (Juno Therapeutic s). In some cases, the CD19-specific CAR-T cells have a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; CD27, CD28, 4-1BB (CD137 ), ICOS, DAP10, OX40 (CD134), or one or more co-stimulatory domains selected from fragments or combinations thereof; and a signaling domain derived from CD3 zeta. It further includes a transmission domain.

[0207] In some embodiments, the CD19-specific CAR-T cells described herein include the anti-CD19 antibody described in US Patent Application Publication No. 20160152723 . It contains.

[0208] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharm a, Inc.). In some In embodiments, as used herein, antigen-binding domains that recognize epitopes on CD19 recognized also by KTE-C19 are described, and CD19-specific CAR-T cells are described. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of an scFv, and the antigen-binding domain recognizes an epitope on CD19 recognized also by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof. or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of an scFv, and the antigen-binding domain recognizes an epitope on CD19 recognized also by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof.

[0209] In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of an scFv, and the antigen-binding domain recognizes an epitope on CD19 recognized also by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof. In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of an scFv, and the antigen-binding domain recognizes an epitope on CD19 recognized also by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof. In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of an scFv, and the antigen-binding domain recognizes an epitope on CD19 recognized also by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof. In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of an scFv, and the antigen-binding domain recognizes an epitope on CD19 recognized also by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof.

[0210] In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof. In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in International Publication No. WO 2015 / 187528, or a fragment or derivative thereof.

[0211] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). In some embodiments, the present specification describes CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In the present specification, CD19-specific CAR-T cells are described in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0212] In some embodiments, the CD19-specific CAR-T cells described herein comprise the antigen-binding domain of scFv, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the CD19-specific CAR-T cells described herein comprise the antigen-binding domain of scFv, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta. In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellestis). In some embodiments, the present specification describes CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some embodiments, the present specification describes CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19. CD19-specific CAR-T cells that recognize an epitope on CD19 and are recognized are described. In some cases, the CD19-specific CAR-T cells include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28 , 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD13 4), or fragments or combinations thereof; and further include a signaling domain derived from CD3 zeta.

[0213] In some embodiments, the CD19-specific CAR-T cells described herein include the antigen-binding domain of scFv and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some cases, the CD19-specific CA R-T cells include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain ; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DA P10, DAP12, OX40 (CD134), or fragments or combinations thereof; and further include a signaling domain derived from CD3 zeta.

[0214] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum). In some embodiments, this specification describes CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD1 9 that is also recognized by BPX-401. In some cases, CD19-specific CAR-T cells are described in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. The CD19-specific CAR-T cells include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3ζ. transmembrane domain; CD27, CD28, 4-1BB (CD 137), ICOS, DAP10, DAP12, OX40 (CD134), or one or more co-stimulatory domains selected from fragments or combinations thereof; and CD3ζ further includes a signaling domain derived from.

[0215] In some embodiments, the CD19-specific CAR-T cells described herein include an antigen-binding domain of scF v that recognizes an epitope on CD19 that is also recognized by BPX-401. In some cases, the CD19-specific CA R-T cells include a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3ζ transmembrane domain; CD27, CD28, 4-1BB (CD137), ICOS, DA selected from the transmembrane domain; CD27, CD28, 4-1BB (CD137), ICOS, DA re; CD27, CD28, 4-1BB (CD137), ICOS, DA P10, DAP12, OX40 (CD134), or one or more co-stimulatory domains selected from fragments or combinations thereof; and a signaling domain derived from CD3ζ. main.

[0216] In some cases, the antigen-binding domain is blinatumomab (Amgen), cortsimab blabtansine (ImmunoGen Inc. / Sanofi-aventis), M OR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetic s), B4 (or DI-B4) (Merck Serono), taplitumomab papto x (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex) or also recognizes an epitope on CD19 that is also recognized by AFM11 (Affimed). In some cases, the CD19-specific CAR comprises a transmembrane domain selected from the CD8 alpha transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and also further comprises a signaling domain derived from CD3ζ. Some embodiments described herein include CD19-specific CAR-T cells wherein the antigen-binding domain comprises F(ab’)2, F ab’, Fab, Fv, or scFv. In some cases, the antigen-binding domain recognizes an epitope on CD19. Optionally, the antigen-binding domain is a domain that recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR20 8 (Morphosys AG / Xencor Inc.), MEDI-551 (Med

[0217] immune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox ins (National Cancer Institute), XmAb 5871 (A mgen / Xencor, Inc.), MDX-1342 (Medarex) or AF M11 (Affimed). 8 (Morphosys AG / Xencor Inc.), MEDI-551 (Med immune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox ins (National Cancer Institute), XmAb 5871 (A mgen / Xencor, Inc.), MDX-1342 (Medarex) or AF M11 (Affimed) that also recognizes an epitope on CD19. It is. In some cases, the CD19-specific CAR-T cells are transmembrane domains selected from the CD8α transmembrane domain or the CD3ζ transmembrane domain; CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or one or more co-stimulatory domains selected from these fragments or combinations; and further include a signaling domain derived from CD3ζ.

[0218] In some cases, the CD19-specific CAR-T cells described herein include the antigen-binding domain of the scFv, and the antigen-binding domain is blinatumomab (Amgen), col tuximab-labatansine (ImmunoGen Inc. / Sanofi-avent is), MOR208 (Morphosys AG / Xencor Inc.), MED I-551 (Medimmune), denintuzumab mafodotin (Seattle G enetics), B4 (or DI-B4) (Merck Serono), taplit umumab paptox (National Cancer Institute), Xm Ab 5871 (Amgen / Xencor, Inc.), MDX-1342 (Meda rex) or AFM11 (Affimed), and recognize an epitope on CD19. In some cases, the CD19-specific CAR-T cells are transmembrane domains selected from the CD8 alpha membrane transmembrane domain or the CD3ζ transmembrane domain; CD 27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX 40 (CD134), or one or more selected from these fragments or combinations ; a co-stimulatory domain; and further comprising a signaling domain derived from CD3ζ.

[0219] In certain embodiments, the polynucleotide encoding the CAR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, selected from the list consisting of SEQ ID NO: 169 (encoding CD1 9 - CD3ζ CAR), SEQ ID NO: 171 (encoding CD19 - CD137 - CD3 ζ CAR), SEQ ID NO: 173 (encoding CD19 - CD28 - CD3ζ CAR ), and SEQ ID NO: 175 (encoding CD19 - CD28 - CDζ CAR further comprising an IgG4 Fc spacer) and comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequences. In certain embodiments, the amino acid sequence comprising the CAR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 1 (CD19 - CD3ζ CAR), SEQ ID NO: 172 (CD19 - CD137 - CD3ζ CAR), SEQ ID NO: 174 (CD19 - CD28 - CD3ζ CAR), and SEQ ID NO: 176 (CD19 - CD28 - CD3ζ CAR further comprising an IgG4 Fc spacer) selected from the list consisting of and has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 1 00% identity thereto.

[0220] In some embodiments, the polynucleotides disclosed herein can comprise a codon - optimized cDNA sequence encoding an anti - CD19 CAR and a cleavable T2A linker that is connected to a cell surface polypeptide (e.g., HER1t). For example, cytotoxic T lymphocytes are engineered to fuse to a cytoplasmic co - stimulatory (CD28) domain that is fused to a cytoplasmic CD3 - ζ domain It is possible to express a CD19-specific chimeric antigen receptor (CAR) that transmits signals via the main body. This polypeptide can further incorporate an extracellular cell tag, for example, in some embodiments, truncated human HER1 (HER1t), truncated type CD20 (CD20t), truncated type CD52 (CD52t), or truncated type LNGFR (LNGFRt), following a C-terminal 2A cleavable linker. In other embodiments, the polypeptide can include a polypeptide construct that includes a truncated variant that precedes a CD19-specific CAR (e.g., linked via a P2A cleavable linker). In other embodiments, the polypeptide can include a polypeptide construct that includes a truncated variant that precedes a CD19-specific CAR (e.g., linked via a P2A cleavable linker). This disclosure presents polynucleotides and polypeptides encoding the CAR and any polypeptide constructs described herein. In certain embodiments, an anti-CD19 CA R polypeptide construct incorporating a cleavable T2A linker and a cell surface polypeptide is at least

[0221] 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from the list consisting of SEQ ID NO: 181 (CD19-CD28-CD3ζ CAR.P2A.Ig kappa signal peptide.HER1t1 by SEQ ID NO: 56) and SEQ ID NO: 185 (CD19-C D137-CD3ζ CAR.E2A.Ig kappa signal peptide.HER1t7 by SEQ ID NO: 68) and is encoded by a polynucleotide comprising a nucleotide sequence having such identity. In certain embodiments, an anti-CD19 CAR polypeptide construct incorporating a cleavable T2A linker and a cell surface polypeptide is SEQ ID NO: 179 (CD19-CD137-C D137-CD3ζ CAR.E2A.Ig kappa signal peptide.HER1t7 by SEQ ID NO: 68) and is encoded by a polynucleotide comprising a nucleotide sequence having such identity. 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from the list consisting of SEQ ID NO: 181 (CD19-CD28-CD3ζ CAR.P2A.Ig kappa signal peptide.HER1t1 by SEQ ID NO: 56) and SEQ ID NO: 185 (CD19-C

[0222] In certain embodiments, an anti-CD19 CAR polypeptide construct incorporating a cleavable T2A linker and a cell surface polypeptide is SEQ ID NO: 179 (CD19-CD137-C D137-CD3ζ CAR.E2A.Ig kappa signal peptide.HER1t7 by SEQ ID NO: 68) D3ζ CAR.T2A.Ig kappa signal peptide.HER1t according to SEQ ID NO: 55 ), SEQ ID NO: 180 (CD19-CD137-CD3ζ CAR.T2A.Ig kappa signal peptide.HER1t according to SEQ ID NO: 55), SEQ ID NO: 182 (CD19-CD2 ...

Claims

**Claim 1** (a) (i) A HER1 domain III consisting of an amino acid sequence having at least 96% identity with the sequence of SEQ ID NO: 200; and (ii) A truncated HER1 domain IV consisting of an amino acid sequence having at least 96% identity with any one of the sequences of SEQ ID NOs: 203 - 209: A cell surface polypeptide; and **(b) A transmembrane domain capable of inducing dimerization of the cell surface polypeptide:** A fusion protein comprising the above. **Claim 2** **(a) The HER1 domain III consists of an amino acid sequence having at least 97% identity with the sequence of SEQ ID NO: 200; and** **(b) The truncated HER1 domain IV consists of an amino acid sequence having at least 97% identity with any one of the sequences of SEQ ID NOs: 203 - 209,** The fusion protein according to Claim 1. **Claim 3** **(a) The HER1 domain III consists of an amino acid sequence having at least 98% identity with the sequence of SEQ ID NO: 200; and** **(b) The truncated HER1 domain IV consists of an amino acid sequence having at least 98% identity with any one of the sequences of SEQ ID NOs: 203 - 209,** The fusion protein according to Claim 1. **Claim 4** **(a) The HER1 domain III consists of an amino acid sequence having at least 99% identity with the sequence of SEQ ID NO: 200; and** **(b) The truncated HER1 domain IV consists of an amino acid sequence having at least 99% identity with any one of the sequences of SEQ ID NOs: 203 - 209,** The fusion protein according to Claim 1. **Claim 5** **(a) The HER1 domain III consists of an amino acid sequence having at least 99.5% identity with the sequence of SEQ ID NO: 200; and** **(b) The truncated HER1 domain IV consists of an amino acid sequence having at least 99.5% identity with any one of the sequences of SEQ ID NOs: 203 - 209,** The fusion protein according to Claim 1. **Claim 6** **(a) The HER1 domain III consists of the amino acid sequence of SEQ ID NO: 200; and** **(b) The truncated HER1 domain IV consists of any one of the amino acid sequences of SEQ ID NOs: 203 - 209,** The fusion protein according to Claim 1. **Claim 7** The fusion protein according to Claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 96% identity with any one of the sequences of SEQ ID NOs: 211 - 217. **Claim 8**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 97% identity with any one of the sequences of SEQ ID NOs: 211 to 217. **Claim 9**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 98% identity with any one of the sequences of SEQ ID NOs: 211 to 217. **Claim 10**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 99% identity with any one of the sequences of SEQ ID NOs: 211 to 217. **Claim 11**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 99.5% identity with any one of the sequences of SEQ ID NOs: 211 to 217. **Claim 12**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of any one of the amino acid sequences of SEQ ID NOs: 211 to 217. **Claim 13**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 96% identity with the sequence of SEQ ID NO:

211. **Claim 14**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 97% identity with the sequence of SEQ ID NO:

211. **Claim 15**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 98% identity with the sequence of SEQ ID NO:

211. **Claim 16**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 99% identity with the sequence of SEQ ID NO:

211. **Claim 17**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of an amino acid sequence having at least 99.5% identity with the sequence of SEQ ID NO:

211. **Claim 18**: The fusion protein according to claim 1, wherein the cell surface polypeptide consists of the amino acid sequence of SEQ ID NO:

211. **Claim 19** **Claim 20**: The fusion protein according to claim 1, wherein the transmembrane domain can form a homodimer containing a complementary dimerization domain. **Claim 21** **Claim 22**: The fusion protein according to claim 1, wherein the transmembrane domain can form a heterodimer together with a complementary dimerization domain. **Claim 23** The fusion protein according to claim 1, wherein the transmembrane domain contains at least one cysteine residue.

22. The fusion protein according to claim 1, wherein the transmembrane domain comprises a glycophorin A transmembrane domain; a glycophorin A-integrin β3 chimeric transmembrane domain; or a CD3 zeta transmembrane domain.

23. The fusion protein according to claim 1, wherein the transmembrane domain is a CD28 transmembrane domain.

24. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:

36.

25. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

36.

26. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

36.

27. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:

36.

28. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO:

36.

29. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO:

36.

30. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO:

36.

31. The fusion protein according to claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 99.5% identity with the amino acid sequence of SEQ ID NO:

36.

32. The fusion protein according to claim 1, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

36.

33. The fusion protein according to claim 1, wherein the transmembrane domain consists of the amino acid sequence of SEQ ID NO:

36.

34. The fusion protein according to claim 23, comprising a peptide linker for conjugating a cell surface polypeptide to a CD28 transmembrane domain.

35. The fusion protein according to claim 1, comprising an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:

57.

36. The fusion protein according to claim 1, comprising an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO:

57.

37. The fusion protein according to claim 1, comprising an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO:

57.

38. The fusion protein according to claim 1, comprising an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO:

57.

39. The fusion protein according to claim 1, comprising an amino acid sequence having at least 99.5% identity with the amino acid sequence of SEQ ID NO:

57.

40. The fusion protein according to claim 1, comprising the amino acid sequence of SEQ ID NO:

57.

41. The fusion protein according to claim 1, consisting of the amino acid sequence of SEQ ID NO:

57.

42. A polynucleotide encoding the fusion protein according to any one of claims 1 to 41.

43. A vector comprising the polynucleotide according to claim 42.

44. The vector according to claim 43, which is a lentiviral vector, a retroviral vector, or a non-viral vector.

45. The vector according to claim 43, wherein the vector is a Sleeping Beauty transposon.

46. An engineered cell comprising the polynucleotide according to claim 42.

47. The engineered cell according to claim 46, wherein the cell is a human cell.

48. The engineered cell according to claim 47, wherein the cell is a T cell or an NK cell.

49. The engineered cell according to claim 47, wherein the cell is a T cell.

50. The engineered cell according to claim 46, which further expresses a recombinant cytokine.

51. The engineered cell according to claim 46, which further expresses a fusion protein comprising an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO:

178.

52. The engineered cell according to claim 46, which further expresses a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

53. The engineered cell according to claim 52, which expresses a CAR that binds to CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII, CD123, and / or VEGFR-2.

54. The engineered cell according to claim 53, wherein the CAR binds to at least one of CD19, CD33, ROR1, mesothelin, CD22, and / or MUC-16.

55. A composition for use in treating cancer, comprising the engineered cell according to claim 46. **Claim 56**: The composition according to claim 55, wherein the engineered cells are present in an amount in the range of 10 ４ to 10 ９ cells per kg of body weight of the subject to which the composition is administered.

57. The operating cells are present in an amount in the range of 10 ４ to 10 ７ cells per kg of body weight of the subject to which the composition is administered, the composition according to claim 55.

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