RU2796334C2 - Expression of new cell marks - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions including a polypeptide construct for providing a modified cell, a polynucleotide, an expression vector, a modified immune effector cell for use in adoptive cell therapy, a method for depleting a modified cell, and a method of the treatment of cancer is described. In one embodiment, the polypeptide construct contains truncated non-immunogenic HER1, CD20, CD52, and LNGFR polypeptides.

EFFECT: invention expands the arsenal of tools for use in adoptive cell therapy.

32 cl, 20 dwg, 6 tbl, 4 ex

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under U.S. Provisional Application No. 62/516,639, filed June 7, 2017, which is hereby incorporated by reference in its entirety.

SEQUENCE LIST

[0002] This application contains a sequence listing that is provided electronically in ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on May 30, 2018, is named 50471-708_601_SL.txt and has a size of 464445 bytes.

BACKGROUND OF THE INVENTION

[0003] Various types of cell therapies are promising for the treatment of diseases that are not amenable to proper treatment using conventional pharmaceutical agents. Blood transfusions represented the first type of cell therapy for the treatment of hematological malignancies. Recent advances in cell isolation and induction technologies, as well as gene transfer, have made it possible to genetically modify different types of cells (primary and immortal) for the treatment of various diseases, such as cancer, cardiovascular, dermatological, neurological and ophthalmic diseases. In many cases, it is very important to enrich the genetically modified cell therapy product to achieve the necessary purity to allow expansion of selected cells of therapeutic interest and/or removal of non-genetically modified or other cell types prior to infusion in patients. In addition, adoptive cellular immunotherapy using, for example, cytokines, chimeric antigen receptors (CARs) and T cell receptors (TCRs) has proven to be very promising in terms of successful direct killing of tumor cells. Although this innovative technology is promising, the administration of modified immune cells to individuals with tumors comes with some safety concerns, such as toxicity, tumor lysis, and cytokine release syndrome (i.e., "cytokine storm") in the case of CAR- T cells. In order to take full advantage of the therapeutic potential offered by adoptive T-cell immunotherapy, side effects such as "cytokine storm" must be controlled during therapy.

INCLUSION BY LINK

[0004] All publications, patents, and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually designated for inclusion by reference.

SUMMARY OF THE INVENTION

[0005] The present invention provides polypeptide constructs and polynucleotides that can be expressed in cells to overcome one or more of the above disadvantages.

[0006] A polypeptide construct and a polynucleotide encoding a polypeptide construct are provided, wherein said polypeptide construct contains a truncated version of a naturally occurring polypeptide. According to some embodiments of the present invention, the polypeptide construct may further comprise a transmembrane domain or fragment thereof, a signal peptide, and/or a peptide linker. The present invention also provides modified cells expressing polynucleotides and polypeptide constructs. The modified cells can express the polypeptide constructs on the cell surface, which provides a cellular marker (or "cell tag") that, in some embodiments of the present invention, uniquely identifies the modified cells.

[0007] In addition, the present invention provides a polypeptide construct and a polynucleotide encoding a polypeptide construct, said polypeptide construct comprising a truncated natural polypeptide and a transmembrane domain. According to certain embodiments of the present invention, the truncated version contains an extracellular domain or part thereof and a transmembrane domain or part thereof. According to some embodiments of the present invention, the polypeptide construct may contain a transmembrane domain and a truncated variant derived from different natural proteins. In some cases, a truncated version of the transmembrane domain of the polypeptide construct is a single-pass transmembrane domain. In other cases, the transmembrane domain of the polypeptide construct is a multipass transmembrane domain.

[0008] The present invention provides a polypeptide construct and a polynucleotide encoding a polypeptide construct, said polypeptide construct comprising a dimerization transmembrane domain that can bind a cell surface polypeptide (e.g., a truncated variant hybridized to the transmembrane domain) on the cell surface to a second cell surface polypeptide . In certain embodiments of the present invention, binding of cell surface polypeptides via the transmembrane dimerization domain can enhance the signal derived from cell surface polypeptides compared to the non-dimerized configuration.

[0009] The present invention also provides a polypeptide construct and a polynucleotide encoding the polypeptide construct, said polypeptide construct comprising domains or fragments thereof that are derived from various naturally occurring proteins. According to some embodiments of the present invention, the polypeptide construct described in this application may contain a truncated version of the natural polypeptide, a transmembrane domain, an optional peptide linker connecting the truncated version to the transmembrane domain, and a signal peptide that directs the polypeptide construct to the cell surface. For example, in some embodiments of the invention, the polypeptide construct comprises a truncated variant or fragment thereof that is derived from a naturally occurring protein other than a transmembrane domain or fragment thereof hybridized directly or indirectly to the truncated variant or fragment thereof. In some embodiments of the present invention, a particular domain (eg, extracellular domain) of the polypeptide construct described herein is chimeric and contains amino acid sequences derived from various naturally occurring proteins.

[00010] In some instances, methods and compositions are provided comprising a polypeptide construct comprising a cell surface polypeptide and a dimerization transmembrane domain, wherein said dimerization transmembrane domain induces dimerization of the cell surface polypeptide and the cell surface polypeptide binds a predetermined antibody or variant or fragment thereof. The present invention also provides polynucleotide sequences encoding the polypeptide construct described herein.

[00011] The present invention provides methods and compositions containing cellular labels that include truncated variants of polypeptides such as HER1, CD20, LNGFR, and CD52. In some cases, truncated polypeptide variants do not bind the endogenous receptor. The disclosed truncated non-immunogenic polypeptides can be used as cellular markers, for example, as a cellular marker, a depletion marker, or a killer marker.

[00012] The present invention provides compositions containing modified cells that express the polypeptide constructs or polynucleotides described herein. In some instances, the modified cells further express at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and/or a cytokine.

[00013] According to the present invention, methods are provided for regulating the activity of genetically modified cells in a subject (for example, undergoing immunotherapy), including providing the subject with genetically modified cells encoding a polynucleotide construct disclosed in this application, as well as providing the subject with a predetermined binding partner that binds cells and regulates their activity. Systems and kits for use in these methods are also provided.

[00014] Provided is a polypeptide construct comprising a cell surface polypeptide and a dimerization transmembrane domain, wherein said dimerization transmembrane domain induces dimerization of said cell surface polypeptide, and wherein said cell surface polypeptide binds a predetermined antibody or variant or fragment thereof.

[00015] In some embodiments, said cell surface polypeptide comprises at least one of: a HER1 polypeptide, a LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. In some cases, said cell surface polypeptide contains a HER1 polypeptide, and said HER1 polypeptide contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical the sequence shown in SEQ ID NO: 211, 212, 213, 214, 215, 216 or 217. In some cases, the specified HER1 polypeptide contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 211, 212, 213, 214, 215 , 216 or 217.

[00016] In some embodiments, said cell surface polypeptide comprises a CD20 polypeptide, and said CD20 polypeptide comprises a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In some cases, the specified CD20 polypeptide contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

[00017] In some embodiments, said cell surface polypeptide comprises an LNGFR polypeptide, and said LNGFR polypeptide comprises a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the sequence shown in SEQ ID NO: 156, SEQ ID NO: 158, or SEQ ID NO: 160. In some instances, said cell surface polypeptide contains an LNGFR polypeptide, and said LNGFR polypeptide contains a polypeptide sequence that is at least at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 156, SEQ ID NO: 158, or SEQ ID NO: 160.

[00018] In some cases, said cell surface polypeptide does not bind an endogenous receptor. In some instances, said transmembrane dimerization domain may form either a homodimer or a heterodimer with a complementary dimerization domain. In some cases, said transmembrane dimerization domain contains at least one cysteine residue. In some embodiments, said dimerization transmembrane domain comprises a glycophorin A transmembrane domain or a fragment or variant thereof, a chimeric glycophorin A-integrin transmembrane domain (33 or a fragment or variant thereof, or a CD3-zeta transmembrane domain. In some instances, such a cell surface polypeptide comprises at least HER1 polypeptide.

[00019] In some cases, said polypeptide construct is expressed in the modified cell. In some embodiments of the present invention, said modified cell is an animal cell. In some instances, said animal cell is a human cell. In some embodiments of the present invention, said human cell is a T cell or an NK cell. In some cases, said modified cell further comprises the Sleeping Beauty transposase.

[00020] In some cases, said modified cell additionally expresses at least one additional exogenous polypeptide. In some cases, said modified cell additionally expresses at least one exogenous receptor polypeptide or fragment thereof. In some instances, said modified cell further expresses at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR) and a cytokine. According to some embodiments of the present invention, said modified cell further expresses at least one CAR, wherein said CAR binds at least one of: CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2.

[00021] In some cases, said modified cell additionally expresses at least one recombinant cytokine. In some cases, the specified recombinant cytokine contains at least one of: IL-15, mbIL-15, IL-2, IL-12 and IL-21. In some embodiments of the present invention, said polypeptide construct is encoded by a polynucleotide inserted into said modified cell by genome editing. In some instances, said genome editing involves the use of at least a site-specific serine recombinase system. In some instances, said polypeptide construct contains a linker that hybridizes said cell surface polypeptide to said transmembrane dimerization domain. In some instances, a polypeptide homodimer or heterodimer contains a polypeptide construct.

[00022] Provided is a polynucleotide encoding a polypeptide construct comprising a non-immunogenic cell surface polypeptide hybridized to a transmembrane dimerization domain, said transmembrane dimerization domain inducing dimerization of said cell surface polypeptide. In some embodiments of the present invention, said cell surface polypeptide does not bind an endogenous receptor. In some instances, said cell surface polypeptide does not have any endogenous signaling or transport functions.

[00023] In some cases, the specified polynucleotide contains at least one sequence encoding at least one heterologous gene. In some embodiments of the present invention, the at least one heterologous gene is modulated with an inducible promoter. In some instances, said at least one heterologous gene contains at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. According to some embodiments of the present invention, said at least one heterologous gene contains said CAR, wherein said CAR binds at least one of: CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2.

[00024] According to some variants of implementation of the present invention, the specified at least one heterologous gene contains a cytokine. In some cases, the specified cytokine contains at least one of: IL-15, IL-2, IL-12, IL-21 and a hybrid of IL-15 and IL-15Rα. According to some embodiments of the present invention, said cytokine is in a secreted form. In some cases, said cytokine is in membrane-bound form.

[00025] According to some embodiments of the present invention, said polynucleotide comprises at least one sequence containing a polypeptide linker selected from the group consisting of 2A linker, GSG-2A, GSG liquor (SEQ ID NO: 16), SGSG linker (SEQ ID NO: 18), furinlink, their variants and derivatives. In some embodiments of the present invention, said 2A linker is a p2A linker, a T2A linker, an F2A linker, or an E2A linker.

[00026] In some instances, said polypeptide construct acts as a label for cell enrichment, cell selection, or cell death induction in cells expressing said cell surface molecule. In some instances, said cell surface polypeptide comprises at least one of: a HER1 polypeptide, a LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. According to some embodiments of the present invention, said dimerization transmembrane domain comprises a glycophorin A transmembrane domain or a fragment or variant thereof, a chimeric glycophorin A-integrin β3 transmembrane domain or a fragment or variant thereof, or a CD3 zeta transmembrane domain.

[00027] In some cases, the vector contains a polynucleotide. In some instances, said 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. In some cases, said polynucleotide is inserted into a modified cell by genome editing. In some instances, said genome editing involves the use of at least a site-specific serine recombinase system.

[00028] A method is provided for regulating the activity of genetically modified cells in a subject, comprising: providing said subject with a number of genetically modified cells encoding a polypeptide construct containing a cell surface polypeptide hybridized to a transmembrane dimerization domain, wherein said transmembrane dimerization domain induces dimerization of said cell surface polypeptide and wherein said cell surface polypeptide binds a predetermined binding partner, or variant or fragment thereof; and providing said subject with said predetermined binding partner in an amount sufficient to bind and thereby regulate the activity of said genetically modified cells.

[00029] In some embodiments of the present invention, said cell surface polypeptide is a non-immunogenic polypeptide. In some embodiments of the present invention, said cell surface polypeptide comprises at least one of: a HER1 polypeptide, a LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. In some instances, said cell surface polypeptide does not bind an endogenous receptor. In some instances, said transmembrane dimerization domain may form either a homodimer or a heterodimer with a complementary dimerization domain. According to some embodiments of the present invention, said transmembrane dimerization domain contains at least one cysteine residue. In some instances, said dimerization transmembrane domain comprises a glycophorin A transmembrane domain or fragment or variant thereof, a chimeric glycophorin A-integrin β3 transmembrane domain or fragment or variant thereof, or a CD3 zeta transmembrane domain.

[00030] In some instances, said binding partner includes an antibody or region thereof that binds a cell surface polypeptide. In some embodiments of the present invention, said antibody comprises at least one of a monoclonal antibody, scFv, scFab, diabody, and camelid antibody. In some instances, said antibody includes at least one of: rituximab, cetuximab, alemtuzumab, panitumumab, and necitumumab.

[00031] In some cases, these genetically modified cells contain at least one of: T cells and NK cells. According to some variants of implementation of the present invention, at least one of these genetically modified cells additionally expresses at least one additional exogenous polypeptide. In some instances, at least one of said genetically modified cells additionally expresses at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. In some cases, at least one of said genetically modified cells additionally expresses at least one CAR, and said CAR binds at least one of: CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE -A1, h5T4, PS MA, TAG-72, EGFRvIII, CD123 and VEGF-R2.

[00032] According to some variants of implementation of the present invention, at least one of these genetically modified cells additionally expresses at least one recombinant cytokine. In some cases, the specified recombinant cytokine contains at least one of: IL-15, mbIL-15, IL-2, IL-12 and IL-21. In some instances, said predetermined binding partner is provided in an amount sufficient to cause a reduction in at least one symptom associated with a cytokine storm or a systemic inflammatory response. In some embodiments of the present invention, said predetermined binding partner is provided in an amount sufficient to cause a reduction in at least one symptom associated with tumor lysis syndrome. In some cases, said polypeptide construct is encoded by a polynucleotide inserted into said modified cells by genome editing. In some cases, said genome editing involves the use of at least a site-specific serine recombinase system.

[00033] Provided is a polynucleotide encoding a truncated non-immunogenic CD20 polypeptide that binds an anti-CD20 antibody, wherein said truncated non-immunogenic CD20 polypeptide does not bind an endogenous receptor.

[00034] According to some embodiments of the present invention, said CD20 polypeptide contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence represented in SEQ ID NO: 109. In some cases, said CD20 polypeptide comprises a polypeptide sequence comprising the sequence of SEQ ID NO: 109. In some cases, said CD20 polypeptide binds said anti-CD20 antibody with a binding efficiency that is at least 50%, 60% , 70%, 80% or 90% of the binding efficiency of native CD20. In some embodiments of the invention, said anti-CD20 antibody comprises at least one of: rituximab, cetuximab, tositumomab, veltuzumab, afutuzumab, blontuvetmab, and obinutuzumab.

[00035] Provided is a polynucleotide encoding a truncated non-immunogenic HER1 polypeptide consisting of at least HER1 domain III or a fragment thereof and a truncated HER1 domain IV, wherein said HER1 polypeptide binds an anti-HER1 antibody, and wherein said HER1 polypeptide is expressed in a modified cell.

[00036] According to some embodiments of the present invention, said truncated HER1 IV domain comprises a truncation of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of said HER1 IV domain.

[00037] In some instances, said HER1 truncated domain IV contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 203, 204, 205, 206, 207, 208, or 209. In some instances, said HER1 truncated domain IV contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 203, 204, 205, 206, 207, 208 or 209. In some embodiments of the invention, said HER1 domain III or fragment thereof comprises a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 200. In some cases, the specified domain III of HER1 contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 200.

[00038] In some cases, said polynucleotide further encodes a CD28 transmembrane domain and a peptide linker for linking said HER1 polypeptide to said CD28 transmembrane domain. In some instances, said polynucleotide encodes a polypeptide construct comprising a polypeptide sequence comprising the sequence shown in SEQ ID NO: 57. In some embodiments, said anti-HER1 antibody comprises at least one of: rituximab, cetuximab, futuximab, depatuxizumab, imgatuzumab , laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab, and zalutumumab.

[00039] Provided is a polynucleotide encoding a truncated non-immunogenic CD52 polypeptide that binds an anti-CD52 antibody, wherein said truncated non-immunogenic CD52 polypeptide does not bind an endogenous receptor, and wherein said non-immunogenic CD52 polypeptide is expressed in a modified cell.

[00040] In some instances, said CD52 polypeptide contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 143. In some instances, said CD52 polypeptide contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 143.

[00041] 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 of the present invention, the at least one heterologous gene is modulated with an inducible promoter. In some instances, said at least one heterologous gene contains at least one of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a cytokine. According to some embodiments of the present invention, said at least one heterologous gene contains a CAR, and wherein said CAR binds at least one of: CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE-A1, h5T4 , PS MA, TAG-72, EGFRvIII, CD123 and VEGF-R2.

[00042] According to some variants of implementation of the present invention, the specified at least one heterologous gene contains a cytokine. According to some variants of implementation of the present invention, the specified cytokine contains at least one of: IL-15, IL-2, IL-12, IL-21 and a hybrid of IL-15 and IL-15Rα. In some cases, said cytokine is in a secreted form. In some cases, said cytokine is in membrane bound form.

[00043] According to some variants of implementation of the present invention, the vector contains the specified polynucleotide. In some instances, said vector is a lentiviral vector, a retroviral vector, or a non-viral vector. In some instances, said non-viral vector is a Sleeping Beauty transposon. In some embodiments of the present invention, said polynucleotide is inserted into a modified cell by genome editing. In some instances, said genome editing involves the use of at least a site-specific serine recombinase system. In some cases, the modified cell encodes a polynucleotide. In some cases, the modified cell is a T cell or NK cell. In some cases, a polynucleotide encodes a polypeptide.

[00044] The present invention also provides a method of treating cancer comprising administering to a subject an effective amount of a modified cell containing a polynucleotide. In some embodiments of the present invention, the method further comprises administering at least one binding partner capable of binding to a polypeptide expressed on said modified cell. In some instances, said binding partner is an antibody.

[00045] According to the present invention, there is provided a method for regulating the activity of genetically modified cells in a subject, comprising providing said subject with a number of genetically modified cells encoding a polypeptide construct containing a cell surface polypeptide, which is a chimeric polypeptide containing a first truncated non-immunogenic polypeptide and a second truncated non-immunogenic a polypeptide, wherein said cell surface polypeptide binds at least one predefined binding partner, or variant or fragment thereof; and providing said subject with said predetermined binding partner in an amount sufficient to bind and thereby regulate the activity of said genetically modified cells.

[00046] According to some embodiments of the present invention, said first truncated non-immunogenic polypeptide comprises a fragment or derivative of an EGFR family member. In some instances, said second truncated non-immunogenic polypeptide contains a fragment or derivative of a member of the EGFR family. In some instances, said first truncated non-immunogenic polypeptide comprises an HER1 polypeptide. According to some embodiments of the present invention, said HER1 polypeptide comprises a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 211, 212, 213, 214, 215, 216, or 217. In some embodiments of the present invention, said HER1 polypeptide comprises a polypeptide sequence comprising the sequence shown in SEQ ID NOs: 211, 212, 213, 214, 215, 216, or 217. In some cases, the specified HER1 polypeptide contains a polypeptide sequence containing the sequence presented in SEQ ID NO: 211.

[00047] In some instances, said second truncated non-immunogenic polypeptide comprises at least one of: an HER2 polypeptide, an ErbB3 polypeptide, and an ErbB4 polypeptide. According to some embodiments of the present invention, said polypeptide construct contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101 or SEQ ID NO: 105.

[00048] According to some embodiments of the present invention, said at least one predefined binding partner binds said HER1 polypeptide. In some instances, said at least one predefined binding partner includes at least one of: rituximab, cetuximab, futuximab, depatuxizumab, imgatuzumab, laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab, and zalutumumab. In some instances, said at least one predefined binding partner further comprises a second predefined binding partner that binds said second truncated non-immunogenic polypeptide. In some instances, said second truncated non-immunogenic polypeptide is an HER2 polypeptide and said second predefined binding partner is pertuzumab.

[00049] In some cases, said polypeptide construct further comprises a signal peptide. In some cases, said polypeptide construct further comprises a transmembrane domain. In some embodiments of the present invention, said transmembrane domain comprises a dimerization transmembrane domain. In some instances, said dimerization transmembrane domain comprises a glycophorin A transmembrane domain, a chimeric glycophorin A-integrin β3 transmembrane domain, or a CD3 zeta transmembrane domain. In some instances, said dimerization transmembrane domain contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32. According to some embodiments of the present invention, said transmembrane dimerization domain comprises a polypeptide sequence comprising the sequence shown in SEQ ID NO: 26, SEQ ID NO: 28 , SEQ ID NO: 30 or SEQ ID NO: 32.

[00050] In some instances, said first truncated non-immunogenic polypeptide comprises a CD20 polypeptide. According to some embodiments of the present invention, said CD20 polypeptide contains a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220. In some instances, said CD20 polypeptide contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220. According to In some embodiments of the present invention, said at least one predefined binding partner comprises at least one of: rituximab, tositumomab, veltuzumab, afutuzumab, blontuvetmab, and obinutuzumab.

[00051] A polypeptide construct is provided comprising a truncated non-immunogenic HER1 polypeptide consisting of HER1 domain III and a truncated HER1 domain IV, said HER1 polypeptide binding an anti-HER1 antibody; transmembrane domain of CD28; and a peptide linker for linking said HER1 polypeptide to said CD28 transmembrane domain; wherein said polypeptide construct is expressed in the modified cell.

[00052] According to some embodiments of the present invention, said HER1 domain III comprises a polypeptide sequence containing the sequence shown in SEQ ID NO: 200, and said HER1 domain IV contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 203. In some cases, said CD28 transmembrane domain contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 36, and said peptide linker contains a G4S peptide linker (SEQ ID NO: 221). In some cases, said G4S peptide linker (SEQ ID NO: 221) contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 22. According to some embodiments of the present invention, said polypeptide construct contains a polypeptide sequence containing the sequence shown in SEQ ID NO: :57.

[00053] In some instances, said anti-HER1 antibody includes at least one of: rituximab, cetuximab, futuximab, depatuxizumab, imgatuzumab, laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab, and zalutumumab.

[00054] Provided is a polypeptide construct comprising a truncated non-immunogenic CD20 polypeptide, wherein said CD20 polypeptide binds an anti-CD20 antibody; wherein said polypeptide construct is expressed in the modified cell.

[00055] In some embodiments of the present invention, said polypeptide construct is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the sequence shown in SEQ ID NO: 109. In some cases, said polypeptide construct contains a polypeptide sequence containing the sequence shown in SEQ ID NO: 109. In some cases, said KCD20 antibody contains at least one of: rituximab, tositumomab, veltuzumab, afutuzumab, blontuvetmab, and obinutuzumab.

BRIEF DESCRIPTION OF THE DRAWINGS

[00056] The features of the present invention are detailed in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the present invention are applied, and the accompanying drawings in which:

[00057] FIG. 1 shows the expression levels of the full-length CD20 cell tag (corresponding to SEQ ID NO: 107) and two forms of truncated CD20 (CD20t1, corresponding to SEQ ID NO: 109 and CD20t4, corresponding to SEQ ID NO: 115) in HEK-293T cells, according to the results of detection by flow cytometry using rituximab, compared with a control cell label. Shaded areas indicate mock-transfected control cells, while open areas indicate expression of cell marks from transfected cells. Each transfection was performed in three repetitions.

[00058] FIG. 2 shows co-expression of the CD20t1 cell marker (corresponding to SEQ ID NO: 109; y-axis) and chimeric antigen receptor (CAR; x-axis) in human peripheral blood mononuclear cells (PBMCs) co-transfected with Sleeping Beauty transposon vectors encoding both genes (right panel) compared to non-transfected cells (left panel).

[00059] FIG. 3 shows specific dose-dependent antibody-dependent cellular cytotoxicity (ADCC) (presented as relative induction; y-axis) induced by rituximab in a Jurkat cell line transfected with CD20t-1. Untransfected original Jurkat cells and the non-specific cetuximab antibody showed no activity.

[00060] FIG. 4A is a schematic diagram illustrating embodiments of the polypeptide construct described herein. The top panel shows an embodiment of a polypeptide construct containing a signal peptide for targeting the polypeptide construct to the cell surface, a truncated natural polypeptide, an optional linker, and a transmembrane (TM) domain. The bottom panel illustrates an embodiment of the polypeptide construct expressed in the modified cell described in this application. The polypeptide construct is attached to the cell surface by a transmembrane (TM) domain that is hybridized to a truncated variant oriented to the extracellular space using an optional peptide linker. In the illustrated embodiment, the linker serves as an extension to guide and distance the truncated variant from the cell surface, thus optimizing the binding of the antibody or binding partner (represented by an antibody to the truncated variant) to the epitope of the truncated variant.

[00061] FIG. 4B is a schematic diagram illustrating an embodiment of the polypeptide construct described herein. The truncated version and optional linker of FIG. 4A are replaced by FIG. 4b HER domain III(m) and HER(n) domain IV, where m and n represent any member of the EGFR/HER family, including HER1, HER2, ErbB3, and ErbB4 (i.e., m=1-4 and n=1- 4, but n≠m). In the embodiment shown, HER(m) domain III and HER(n) domain IV each represent different epitopes that are recognized by different antibodies (ie, anti-HER(m) and HER(n) antibodies, respectively).

[00062] FIG. 4C is a schematic diagram illustrating a specific embodiment in which HER(m) domain III in FIG. 4B corresponds to EGFR domain III recognized by an anti-EGFR antibody that hybridizes at the C-terminus to either HER2 or ErbB3 or ErbB4 domain IV followed by a TM domain.

[00063] FIG. 4D shows expression of a truncated HER1 polypeptide (HER1t) expressed in human donor PBMCs transfected with various EGFR-Erb4 chimeras (truncated EGFR-ErbB4 (JM-a) corresponding to SEQ ID NO: 101 and truncated EGFR-ErbB4 (JM-b) corresponding to SEQ ID NO: 105) and stained with an anti-HER1t antibody ("stain") compared to isotype control ("isotype") and control cells that were not transfected with HER1t.

[00064] FIG. 4E shows HER1t polypeptide expression in human donor PBMCs transfected with various HER1t variants (HER1t1 corresponding to SEQ ID NO: 57, HER1t2 corresponding to SEQ ID NO: 59, HER1t3 corresponding to SEQ ID NO: 61, HER1t4 corresponding to SEQ ID NO: 63 , HER1t5 corresponding to SEQ ID NO: 65, HER1t6 corresponding to SEQ ID NO: 67, and HER1t7 corresponding to SEQ ID NO: 69), compared with control cells that were not transfected with HER1t.

[00065] FIG. 5A shows antibody-based detection of HER1t1 expression (corresponding to SEQ ID NO: 57; y-axis) and CAR expression (x-axis) measured by flow cytometry in cells co-transfected with a lentiviral vector encoding both proteins (right panel) compared to control cells not transfected with HER1t or CAR (left panel).

[00066] FIG. 5B shows the levels of HER1t1 (corresponding to SEQ ID NO: 57; y-axis) and CAR (x-axis) in cells transfected with Sleeping Beauty transposon vectors encoding only CAR (left panel) and CAR together with HER1t1 (right panel) and stained with antibody. to HER1 and CAR-specific proteins (lower panel), compared to isotype control (upper panel).

[00067] FIG. 6 shows ADCC activity mediated by cetuximab and alemtuzumab (expressed as % specific kill; y-axis) in NK cells (left), CD16-NK cells (center), and CD16 NK cells transfected with CAR and HER1t1 (right). Cetuximab showed specific ADCC in the presence of HER1t1.

[00068] FIG. 7A shows expression of CAR and HER1t1 cell label (corresponding to SEQ ID NO: 57) measured by flow cytometry in a genetically modified SUP-T1 reporter cell line (SUP-T1/CAR-HER1t1). The SUP-T1/CAR-HER1t1 cell line was sorted by fluorescence activated cell sorting (FACS) to enrich for high (middle panel) or moderate (right panel) HER1t1 expression as determined by flow cytometry detection. The left panel shows SUP-T1/CAR-HER1t1 staining using an isotype control antibody for flow cytometry. FIG. 7B shows specific ADCC (presented as relative induction; y-axis) of the SUP-T1/CAR-HER1t1 cell line induced by cetuximab, which is dependent on HER1t1 expression as well as cetuximab dose levels. The non-specific antibody rituximab did not show SUP-T1/CAR-HER1H ADCC.

[00069] FIG. 8 shows Western blot analysis showing expression of the HER1t1 cell marker in genetically modified SUP-T1 cell lines. Lane 1: i.p. antibody only; Lane 2: Jurkat cages only; Lane 3: SUPT1/HER1t1 (high levels of HER1t1); Lane 4: SUPT1/HER1t1 (low levels of HER1t1); Lane 5: A431 cells expressing full length HER1; Lane 6: marker. The bold arrow indicates the protein affinity precipitated by cetuximab in all lines except A431. HER1t1 corresponds to SEQ ID NO: 57.

[00070] FIG. 9 shows the efficiency of elimination of CD19 CAR-T cells co-expressing the HER1t1 cell label by ADCC (left panel) and complement-dependent cytotoxicity (CDC) (right panel) in the presence of cetuximab or the non-specific rituximab antibody.

[00071] FIG. 10 is a schematic diagram showing various embodiments of the polypeptide constructs described herein. Truncated first generation cell labels (top) contain a truncated variant and a non-dimerizable transmembrane domain (TM) connected by an optional peptide linker. Truncated next generation cell labels (bottom panel) contain a transmembrane dimerization domain n (TM-A) that facilitates dimerization of the truncated variants on the cell surface. In both cases, the antibody to the truncated variant binds to an epitope on the truncated variant.

[00072] FIG. 11 shows CD19 CAR expression levels in primary T cells modified to co-express CD19 CAR and the HER1t cell label. Useful HERtl cell labels include a truncated HER1 polypeptide with a transmembrane dimerization domain (HER1t8 variants corresponding to SEQ ID NO: 71, HER1t9 corresponding to SEQ ID NO: 75, and HER1t10 corresponding to SEQ ID NO: 79) and a polypeptide construct containing the truncated polypeptide HER1 without transmembrane dimerization domain (HER1t1 corresponding to SEQ ID NO: 57) compared to T cells expressing CD19 CAR only (% of baseline).

[00073] FIG. 12 shows expression levels of the HER1t cell label in primary T cells modified to co-express CD19 CAR and the HER1t cell label. Useful HER1t cell markers include a truncated HER1 polypeptide with a transmembrane dimerization domain (HER1t8 variants corresponding to SEQ ID NO: 71, HER1t9 corresponding to SEQ ID NO: 75, and HER1t10 corresponding to SEQ ID NO: 79) and a polypeptide construct containing the truncated polypeptide HER1 without a dimerization domain (HER1t1 corresponding to SEQ ID NO: 57) compared to T cells expressing only the CD19 CAR (% of baseline).

[00074] FIG. 13A and FIG. 13B shows more favorable effects of cetuximab-mediated ADCC (represented as % specific lysis; y-axis) of CD19 CAR-T target cells co-expressing a polypeptide construct containing a truncated HER1 polypeptide with a dimerization domain (HER1t8 variants corresponding to SEQ ID NO: 71, HER1t9 corresponding to SEQ ID NO: 75 and HER1t10 corresponding to SEQ ID NO: 79), compared with a polypeptide construct containing a truncated HER1 polypeptide without a dimerization domain (HER1t control corresponding to SEQ ID NO: 57). NK cells were used as effector cells. Rituximab was used as a control non-specific antibody for the analysis of ADCC.

[00075] FIG. 14 shows the selective elimination of genetically modified T cells (CD19 CAR-mblL-15-T cells) expressing HER1t1 by cetuximab-mediated ADCC.

DETAILED DESCRIPTION

[00076] The following description and examples illustrate embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific implementation options described in this application, and, accordingly, may vary. Those skilled in the art will appreciate that there are many variations and modifications of the present invention that are included within its scope.

[00077] It is understood that all terms correspond to the understanding of a person skilled in the art. Unless otherwise indicated, all technical and scientific terms are used in this application in the meaning corresponding to the usual understanding of a person skilled in the art to which the present invention belongs.

[00078] The section headings used in this application are for organizational purposes only and should not be construed as limiting the described subject matter.

[00079] While various features of the present invention may be described in terms of one embodiment, the features may also be presented singly or in any suitable combination. Conversely, while for clarity, the present invention may be described herein in terms of particular embodiments, the present invention may also be practiced in one embodiment.

[00080] The following definitions are in addition to the definitions in the art and refer to this application and should not be attributed to any related or unrelated matter, for example, any public patent or application. Although any methods and materials similar or equivalent to those described in this application can be used in practice to test the present invention, the present application describes the preferred materials and methods. Accordingly, the terminology used in this application is only intended to describe specific implementations and is not intended to be limiting.

[00081] In this application, the use of the singular includes the plural, unless specifically stated otherwise. It should be noted that in the present description, forms denoting an element in the singular (respectively, "a" or "an" in the original text in English) include plural references, unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or", unless otherwise indicated. In addition, the use of the term "including", as well as other forms such as "include", "includes" and "included", is not limiting.

[00082] Reference in the description to "certain implementations", "an implementation", "one implementation", or "other implementations" means that a particular feature, structure, or characteristic described in connection with the implementations is included at least in some embodiments, but not necessarily all embodiments, of the present invention.

[00083] In this specification and claims, the words "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 “includes” and “include”), or “including” (and any form of “including”, such as “includes” and “enclose”) are inclusive or open and do not exclude additional, not specified elements or steps of the method. It is contemplated that any implementation option discussed in this description can be implemented in relation to any method or composition according to the present invention, and vice versa. In addition, the compositions according to the present invention can be used to achieve the methods according to the present invention.

[00084] As used herein, the term "about" in relation to a reference numeric value and its grammatical equivalents may include the numeric value itself and a range of values plus or minus 10% of that numeric value. For example, the number "about 10" includes 10 and any number from 9 to 11. For example, the term "about" in relation to the reference number can also include a range of plus or minus 10%, 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2% or 1% of this value.

[00085] By "isolated" is meant the extraction of a nucleic acid from its natural environment. By “purified” is meant that the degree of purity of a given nucleic acid, whether extracted from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified in the laboratory, has been increased, with “purity " is a relative term, not "absolute purity." However, it should be understood that nucleic acids and proteins can be prepared with diluents or adjuvants and are still isolated for practical purposes. For example, nucleic acids are typically mixed with a suitable carrier or diluent when used for administration to cells.

[00086] As used herein, the term "polynucleotide" or "oligonucleotide" refers to the polymeric form of nucleotides of any length, both ribonucleotides and deoxyribonucleotides. This term refers only to the primary structure of a molecule. Thus, the term includes double-stranded and single-stranded DNA, triplex DNA, and double-stranded and single-stranded RNA. It also includes modified, eg by methylation and/or capping, and unmodified forms of the polynucleotide. The term is also intended to include molecules that include non-natural or synthetic nucleotides, as well as nucleotide analogs.

[00087] "Polypeptide" is used interchangeably with the terms "polypeptides" and "protein(s)" and refers to a polymer of amino acid residues. "Mature protein" is a protein that is full-length and that optionally includes glycosylation or other modifications typical of protein in a given cellular environment.

[00088] The polypeptides and proteins disclosed in this application (including their functional parts and functional variants) may contain synthetic amino acids in place of one or more naturally occurring 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- and trans-4-hydroxyproline, 4-aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, 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 and α-tert-butyl glycine. The present invention also contemplates that expression of the polypeptides described herein in a modified cell may be associated with post-translational modifications of one or more amino acids of the polypeptide constructs. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidonecarboxylic acid, disulfide bridge formation, sulfation, myristoylation , palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

[00089] As used herein, "antibody" refers to monoclonal or polyclonal antibodies. A complete antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable region (VH) and three C-terminal constant regions (CH1, CH2 and CH3), and each light chain contains one N-terminal variable region (VL) and one C-terminal constant region (CL). The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have a similar overall structure, with each region containing four framework regions whose sequences 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 region" of an antibody, which is responsible for antigen binding.

[00090] An "antigen recognition fragment or domain" refers to a molecule or portion of a molecule that specifically binds to an antigen. In some embodiments of the present invention, the antigen recognition moiety is an antibody, antibody-like molecule, or fragment thereof, and the antigen is a tumor antigen.

[00091] "Antibody-like molecules" can be, for example, proteins that are members of the Ig superfamily that are capable of selectively binding a partner. MHC molecules and T-cell receptors are such molecules. In some embodiments, the antibody-like molecule is a TCR. In some embodiments of the present invention, the TCR has been modified to increase its binding affinity for MHC.

[00092] The terms “antibody fragment,” “antibody fragment,” “functional antibody fragment,” and “antigen-binding portion” are used interchangeably herein to refer to one or more antibody fragments or portions that retain the ability to specifically bind to an antigen (see, in general, Holliger et al., Nat Biotech., 23(9): 1126-1129 (2005)). An antibody fragment desirably contains, for example, one or more CDRs, variable regions (or parts thereof), constant regions (or parts thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab') ₂ fragment, which is a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the stem region; (iii) an Fv fragment consisting of the VL and VH domains of one antibody arm; (iv) single chain Fv (scFv), which is a monovalent molecule consisting of two Fv fragment domains (i.e. VL and VH) connected by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see e.g. , Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol ., 16:778 (1998)), and (v) a diabody which is a dimer of polypeptide chains, each polypeptide chain having a VH connected to a VL by a peptide linker that is too short to allow pairing between VH and VL on the same and of the same polypeptide chain, which facilitates the pairing of complementary domains on different VH-VL polypeptide chains to form a dimeric molecule having two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, for example, US Pat. No. 8,603,950.

[00093] Nucleic acids and/or nucleic acid sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are homologous if the DNA encoding them is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules may be called homologues. For example, any natural proteins described in this application can be modified using any available method of mutagenesis. When expressed, this mutated nucleic acid encodes a polypeptide that is homologous to the protein encoded by the parent nucleic acid. Homology is usually calculated based on sequence identity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of sequence identity that can be used to establish homology varies depending on the nucleic acid and protein in question, but only 25% sequence identity is typically used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, can also be used to establish homology.

[00094] The terms "identical" or "sequence identity", when applied to two nucleic acid sequences or amino acid sequences of polypeptides, refers to residues in two sequences that are the same when aligned for maximum match in a specified comparison window. In some embodiments, the polypeptide in this application is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or fragment thereof. , for example, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to the original nucleic acid, for example, they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100 % are identical to the reference nucleic acid or its fragment, for example, as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage of sequence identity with a larger molecule, this means that when two molecules are optimally aligned, the specified percentage of residues in the smaller molecule matches the residues in the larger molecule according to the order in which the two molecules are optimally aligned.

[00095] A "transposon" or "transposable element" (TE) is a vector DNA sequence that can change its position in the genome, sometimes creating or reversing mutations and changing the size of a cell's genome. Transposition often results in TE duplication. Class I TEs are copied in two steps: first, they are transcribed from DNA to RNA, and then the resulting RNA is reverse-transcribed into DNA. This copied DNA is then inserted at a new position in the genome. The reverse transcription step is catalyzed by reverse transcriptase, which can be encoded by the TE itself. The characteristics of retrotransposons are similar to those of retroviruses such as HIV. The cut-and-paste transposition mechanism of class II TEs does not include intermediate RNA. Transpositions are catalyzed by several transposase enzymes. Some transposases bind non-specifically to any target site in DNA, while others bind to specific target DNA sequences. Transposase makes a staggered cut at the target site resulting in single-stranded 5- or 3-stranded DNA overhangs (sticky ends). This step cuts out the DNA transposon, which is then ligated into a new target site; this process includes the activity of DNA polymerase, which fills in the gaps, and DNA ligase, which closes the sugar phosphate backbone. This results in duplication of the target site. Insertion sites of DNA transposons can be identified by short direct repeats, which can be created by stepwise cutting in the target DNA and filling with DNA polymerase, followed by a series of inverted repeats important for transposase excision of TEs. TEs using the "cut and paste" mechanism can duplicate if their transposition occurs during the S-phase of the cell cycle, when the donor site is already replicated, but the target site is not yet replicated. Transposition can be classified as "autonomous" or "non-autonomous" in both class I and class II TEs. Autonomous TEs can move on their own, while non-autonomous TEs need another TE to move. This is often because nonautonomous TEs lack transposase (for class II) or reverse transcriptase (for class I).

[00096] "Transposase" refers to an enzyme that binds to the end of a transposon and catalyzes the transposition of the transposon to another part of the genome by a cut and paste or replication transposition mechanism. According to some variants of implementation of the present invention, the catalytic activity of transposase can be used to move the gene (s) from the vector into the genome.

[00097] The nucleic acid sequences and vectors disclosed or provided herein may be introduced into a cell by "transfection", "transformation", "nucleofection", or "transduction". As used herein, "transfection", "transformation" or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical means. Many transfection techniques are known in the art and include, for example, DNA coprecipitation with calcium phosphate (see, for example, Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome mediated transfection; tungsten-assisted microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and coprecipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)); and nucleofection (Trompeter et al., J. Immunol. Methods 274:245-256 (2003). Phage or viral vectors can be introduced into host cells after propagation of infectious particles in suitable packaging cells, many of which are commercially available.

[00098] As used herein, "tumor antigen" refers to any antigenic substance produced or overexpressed in tumor cells. For example, it can trigger an immune response in the host. Alternatively, for the purposes of the present invention, tumor antigens may be proteins that are expressed by both healthy and tumor cells, but because they identify a particular type of tumor, they are a suitable therapeutic target.

[00099] "Promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. The promoters are located near the start sites of gene transcription, on the same strand and in the 5' direction from DNA (in the direction of the 5' region of the sense strand). Some promoters are constitutive because they are active in the cell under all circumstances, while others are regulative because they become active in response to specific stimuli, such as an inducible promoter.

[000100] The term "promoter activity" refers to the degree of expression of a nucleotide sequence that is operably linked to a promoter whose activity is being measured. Promoter activity can be measured directly by quantifying the resulting RNA transcript, such as by Northern blotting, or indirectly by quantifying the product encoded by an attached nucleic acid sequence, such as a reporter nucleic acid sequence, coupled to a promoter.

[000101] As used herein, "inducible promoter" refers to a promoter whose activity is induced in the presence or absence of transcriptional regulators, such as biotic or abiotic factors. Inducible promoters are suitable because the expression of genes operably linked to them can be turned on or off at certain developmental stages in an organism or in a particular 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 of the present invention, the inducible promoter is part of a genetic switch.

[000102] As used herein, the term "enhancer" refers to a DNA sequence that increases transcription, such as the nucleic acid sequence to which it is operably linked. Enhancers can be located many thousands of base pairs away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation profiles, or changes in DNA structure. A large number of enhancers from various sources are well known in the art and are available as or within cloned polynucleotides (eg, from repositories such as ATCC, as well as from other commercial or personal sources). A number of polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located in the 5' direction, within or in the 3' direction relative to the coding sequences. The term "Ig enhancers" refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include, for example, heavy chain (mu) 5' enhancers, light chain (kappa) 5' enhancers, kappa and mu intron enhancers and 3' enhancers (outline Paul W. E. (ed), Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pages 353-363; and patent USA No. 5885827).

[000103] As used herein, "coding sequence" refers to a segment of a polynucleotide that encodes a polypeptide. The region or sequence is limited by a start codon closer to the 5' end and a stop codon closer to the 3' end. Coding sequences may also be referred to as open reading frames.

[000104] As used herein, "operably linked" refers to the physical and/or functional connection of a DNA segment to another DNA segment so as to allow the segments to function in their intended ways. A DNA sequence encoding a gene product is operably linked to a regulatory sequence if it is linked to a regulatory sequence, such as, for example, promoters, enhancers and/or silencers, in a manner that modulates the transcription of the DNA sequence, either directly or indirectly. For example, a DNA sequence is operably linked to a promoter if it is ligated to the promoter in the 3' direction relative to the transcription start site of the promoter, in the correct reading frame relative to the transcriptional start site, and allows for the process of prolonging transcription along the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product if it is ligated to the DNA sequence in such a way as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers can be located in the 5'-direction, in the 3'-direction relative to the coding regions of the DNA sequence, or may be built into them. The signal sequence DNA is operably linked to the DNA encoding the polypeptide if the signal sequence is expressed as a pre-protein that is involved in the secretion of the polypeptide. Linking DNA sequences to regulatory sequences is generally accomplished by ligation at suitable restriction sites or by adapters or linkers inserted into the sequence using restriction endonucleases known to those skilled in the art.

[000105] The term "transcriptional regulator" refers to a biochemical element whose function is to prevent or inhibit transcription of a DNA sequence driven by a promoter under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to promote or stimulate transcription of a DNA sequence , driven by the promoter, under certain environmental conditions (for example, an inductor or enhancer).

[000106] The term "induction" refers to an increase in transcription of a nucleic acid sequence, promoter activity, and/or expression caused by a transcriptional regulator, relative to some initial level of transcription.

[000107] A "target" gene or "heterologous" gene or "gene of interest (GOI)" refers to a gene introduced into a host cell by gene transfer.

[000108] As used herein, "recombinase" refers to a group of enzymes that can facilitate site-specific recombination between specific sites, where the sites are physically separated on a single DNA molecule or the sites are on separate DNA molecules. The DNA sequences of certain recombination sites are not necessarily identical. The initiation of recombination depends on the protein-DNA interaction, the group contains a large number of proteins that catalyze the integration and excision of the phage (for example, λ-integrase, fC31), separation of circular plasmids (for example, Tn3, gamma-delta, Cre, Flp), inversion DNA for alternating gene expression (eg Hin, Gin, Pin), developmental gene assembly (eg Anabaena nitrogen fixation genes), and transposition (eg IS607 transposon). Most site-specific recombinases belong to one of two families based on evolutionary and mechanistic relationships. These include the λ-integrase or tyrosine recombinase family (eg Cre, Flp, Xer D) and the resolvase/integrase or serine recombinase family (eg fC31, TP901-1, Tn3, gamma-delta).

[000109] "Recombination attachment sites" are specific polynucleotide sequences that are recognized by the recombinase enzymes described herein. Typically, two different sites (called "complementary sites") are involved, one of which is present in the target nucleic acid (e.g., in a eukaryotic or prokaryotic chromosome or episome) and the other is present in the nucleic acid to be integrated into the target site. recombination. In this application, the terms "attB" and "attP" are used, which refer to attachment (or recombination) sites derived from a bacterial target and a donor phage, respectively, although the recombination sites for specific enzymes may have different names. Recombination sites typically include the left and right shoulders separated by a cow or spacer area. Thus, the attB recombination site consists of BOB', where B and B' are the left and right arms, respectively, and O is the core region. Similarly, attP is POP', where P and P' are arms and O is also the core region. After recombination between the attB and attP sites and the concomitant integration of the nucleic acid into the target, the recombination sites that flank the integrated DNA are called "attL" and "attR". The attL and attR sites, using the terminology above, are thus composed of BOP' and ROB'', respectively. In some representations in this application, "O" is omitted, and attB and attP, for example, are designated as BB' and PP', respectively.

[000110] The term "gene editing" or "genome editing" refers to the insertion, deletion or replacement of DNA nucleotides in the genome of a living organism. Typically, genome editing uses modified nucleases that can create site-specific double-strand breaks at a predetermined location in the genome. The present invention provides for any means for editing genomes. Non-limiting examples of genome editing techniques include CRISPR, Argonaute, and the AttSite site-specific serine recombinase systems. As used herein, "CRISPR gene editing system" of "CRISPR system" refers to any RNA-guided and Cas protein-mediated process for targeting a change in a DNA sequence to a specific region of the genome. As used herein, "Argonaute gene editing system" refers to any single-stranded DNA directed and Argonaute endonuclease mediated process for targeting a change in a DNA sequence to a specific region of the genome. As used herein, "AttSite gene editing system" or "site-specific serine recombinase gene editing system" or "site-specific serine recombinase system" refers to any process that includes providing a eukaryotic cell that contains a first recombination attachment site and a second recombination attachment site; bringing the first and second recombination attachment sites into contact with a prokaryotic recombinase polypeptide, which results in recombination between the recombination attachment sites, wherein said recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, wherein the first recombination attachment site is a recombination attachment site of a phage genome (attP) or a bacterial genome recombination site (attB), the second recombination site is attB or attP, and the recombinase is selected from the group consisting of Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase and Mycobacterium smegmatis phage recombinase, provided that if the first recombination attachment site is attB, the second recombination attachment site is attP, and if the first recombination attachment site is attP, the second recombination attachment site is attB. Exemplary embodiments of the AttSite serine recombinase system are provided in US Pat. No. 9,034,650, which is incorporated herein by reference.

[000111] As used herein, the term "endogenous" when applied to a molecule, such as a polynucleotide or polypeptide, refers to the natural form of the molecule that can be found in a wild-type cell or organism. A molecule that is found endogenously in the body can be contrasted with a modified molecule described in this application that is not normally found in nature. For example, the modified molecule may contain a variant of a naturally occurring polypeptide or polynucleotide. In some embodiments of the present invention, the natural polypeptide variant is a truncated version of the natural polypeptide. As used herein, the term "truncated variant" refers to a protein or polypeptide lacking one or more amino acid sequences and/or domains relative to an endogenous variant of the protein or polypeptide. For example, a truncated variant inserted into a polypeptide construct described herein may lack an amino acid sequence that corresponds to a domain (eg, intracellular signaling domain, transmembrane domain, ligand-binding domain, etc.) normally present in an endogenous protein. The natural variant of the truncated polypeptide can be derived from any organism, including mammalian species such as mice, rats, rabbits and humans. The modified polynucleotides and polypeptides described in this application can be expressed in a modified cell. In the present application, a modified cell is a cell that has been modified from its natural or endogenous state. An example of a modified cell is a cell described herein that has been modified (eg, by transfection of a polynucleotide into the cell) to encode a truncated natural polypeptide or a truncated natural polynucleotide.

Polypeptide constructs

[000112] This application discloses polypeptide constructs, polynucleotides encoding them, modified cells carrying and/or expressing polypeptide constructs and polynucleotides, and methods for regulating the activity of the modified cells. The modified cells described herein may include immune effector cells modified to encode and express cytokines, chimeric antigen receptors, and T cell receptors.

[000113] In this application, the term "regulation" or "regulation", in relation to modified cells or polypeptide constructs expressed in them, generally refers to the regulation of the activity or number of modified cells after administration to a subject. In some embodiments of the present invention, regulation of the activity of modified cells refers to the depletion (depletion) of modified cells in a subject. According to some embodiments of the present invention, regulation of the activity of modified cells refers to the depletion of some of the modified cells in a subject by administering to the subject an amount of an antibody or binding partner that binds a polypeptide construct expressed on the modified cells. According to some embodiments of the present invention, regulation of the activity of modified cells refers to the depletion of modified cells by activating cell death by binding an antibody or binding partner to a polypeptide construct described herein expressed or associated with said modified cell. In some embodiments of the present invention, regulation of the activity of modified cells refers to activation of the ADCC or CDC pathway in modified cells.

[000114] The polypeptides, polynucleotides and modified cells disclosed in this application, together represent a set of therapeutic agents that can be used to improve the effectiveness of conventional types of immunotherapies and, in particular, adoptive cell therapy. For example, the modified cells described herein can encode and express the polypeptide construct on the cell surface as a new cell label. According to some embodiments of the present invention, the cell label can function as a cell marker that labels, labels, or marks a cell expressing the cell label as a modified cell. In embodiments in which a cell label has been modified to eliminate epitopes recognized by endogenous proteins, such cell label can function to uniquely differentiate the modified cell from other cells in the body, such as during adoptive cellular immunotherapy.

[000115] In other examples, the polypeptides, polynucleotides, and modified cells described herein can be used to minimize or eliminate various immunotherapy toxicities in subjects where safety is a concern (for example, due to the likelihood of a cytokine storm). The cellular markers described herein may contain one or more epitopes recognized by the antibody introduced during immunotherapy to induce a cellular mechanism that will slow down the therapeutic outcome and/or reduce the potential side effects of the therapy. In some embodiments of the present invention, an antibody binds to an epitope to induce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CCC). Accordingly, the cell labels described herein can provide a marker of depletion or "mark to kill" unique to modified cells, allowing practitioners to monitor therapeutic outcome and thereby optimize the efficacy and safety of therapy.

[000116] The immunotherapeutic arsenal described herein enhances conventional immunotherapy by enabling the control of adoptive cellular therapeutic interventions. In some embodiments of the present invention, modified cells can be sensitized to cell depletion strategies by expressing a polypeptide construct capable of dimerizing or multimerizing on the surface of the modified cells. Such dimerized or multimerized polypeptides can be used to enhance the depletion signal and thereby rapidly suppress or eliminate modified immune cell therapy in subjects who are prone to or experience therapy-related side effects. The introduction of modified cells expressing a dimerizable or multimerizable polypeptide construct can provide additional control and optimization of cell depleting interventions. According to additional implementation options, the practical use of the polynucleotide constructs disclosed in this application, as part of a "switch to kill" (or "switch to suicide") system that uses an inducible promoter, can provide control over the moment of expression of a particular polypeptide construct, which creates additional immunotherapy checkpoints at both transcriptional and post-translational levels.

[000117] In another embodiment, the cell labels described herein can be used to enrich modified cells that specifically express such cell labels. For example, cell labels can be used to enrich certain modified cells in order to isolate certain modified cells that express only such cell labels to achieve the required purity to allow propagation of selected cells of therapeutic interest. If necessary, various methods can be used, such as FACS, column purification or magnetic bead methods.

[000118] The polypeptide constructs disclosed in this application may contain one or more domains or specific fragments thereof. Typically, a polypeptide construct may comprise a signal peptide sequence, an extracellular domain, a peptide linker, and a transmembrane domain, each of which functions to confer a specific target property to the polypeptide construct. For example, the polypeptide construct may contain a signal peptide for post-translational targeting of the polypeptide construct to the cell surface; a transmembrane domain for attaching a polypeptide domain to a cell; an extracellular portion, which may contain a truncated version of the natural polypeptide; and a peptide linker connecting the transmembrane domain to the extracellular portion. According to some embodiments of the present invention, the peptide linker functions as an extracellular peptide extension to locate the extracellular portion of the polypeptide in the extracellular matrix and thereby make it available for conferring cell label functionality (e.g., to present or extend an epitope to allow access for antibody binding). In certain embodiments of the present invention, one or more of the above domains may not be present in the polypeptide construct. For example, the polypeptide construct described in this application can be modified so as not to contain a peptide linker domain. In other embodiments, the polypeptide construct contains one or more domains derived from different proteins (ie, the polypeptide construct is chimeric).

[000119] In this application, the term "extracellular", in relation to part of the polypeptide design, refers to the amino acids of the polypeptide, which are located on the outside of the cell membrane. In some embodiments of the present invention, the extracellular portion may be referred to as a cell surface polypeptide. Typically, a portion (eg, distal portion) of the cell surface polypeptide protrudes or protrudes distally into the extracellular space relative to the plasma membrane of the cell. In some instances, the projection may be bound to and therefore recognized by an antibody or antigen recognition polypeptide that has a structure that is complementary and specific to that of the projection. A cell surface polypeptide may include polypeptides or fragments thereof that occur naturally on the cell surface, as well as polypeptides or fragments thereof that are not naturally found on the cell surface (eg, a truncated version of a naturally occurring polypeptide).

[000120] The extracellular portion or cell surface polypeptide may include a truncated version of the naturally occurring polypeptide. The truncated variant may, for example, protrude from the outside of the plasma membrane (eg, via a linker connected to or adjacent to the transmembrane domain) into the extracellular space. According to certain embodiments of the present invention, the truncated version lacks amino acids that are part of the intracellular domain and/or the transmembrane domain in the natural version of the polypeptide. The truncated variant may be modified from the endogenous polypeptide in any way to obtain the extracellular portion of the cell label. For example, the truncated version can be modified to restore or eliminate the amino acids that normally constitute an epitope in the extracellular domain that can be recognized by an endogenous protein such as an antigen or receptor. By removing amino acids that normally interact (eg, in cellular signaling pathways) with extracellular molecules, the truncated variant can be rendered non-reactive or immunologically/epitopically silent at the cell surface, or reduced or minimized in its reactivity or binding to endogenous molecules. Any modification of a natural polypeptide to remove natural epitopes is contemplated herein, including truncation of all or part of the extracellular domain and removal of one or more amino acids that form part of an epitope or are post-translationally modified to form part of an epitope.

[000121] Although the truncated version described in this application may be epitopically silent with respect to endogenous signaling pathways, the truncated version may contain one or more epitopes that can be recognized by a molecule (for example, an antibody) that would not normally enter in contact with the surface of the cell corresponding to the modified cell described in this application. In some embodiments, an antibody or binding partner specific for the epitope of the truncated variant is administered exogenously (eg, during immunotherapy using adopted cells). In this regard, an epitope of a truncated variant that can be recognized by the introduced antibody or fragment thereof may be referred to as inactive or latent. Thus, a cell label inserted into the cell surface of a modified cell used for adoptive immunotherapy may contain an epitope that is inactive until the appropriate molecule is administered to the subject to trigger or activate the cell label (i.e., by epitope recognition). Such an epitope does not interfere with the therapeutic outcome of the modified cells if therapy is continued as intended (i.e., the epitope remains silent or latent), but contains a trigger that can be activated to suppress the cellular outcome if, for any reason, immunotherapy must be suppressed. The present invention contemplates the use of any antibody or small molecule that is capable of recognizing an epitope of a cell label to suppress the therapeutic outcome of a modified cell expressing the label by eliminating such cells (eg, by ADCC or CDC). Unlimited examples of antibodies, which can be used to recognize the epitope of the extracellular domain (for example, truncated version) of the cell mark, include rituximab, cetuximab, gephitinib, erlotinib, apatinib, brigatinib, icotinib, icotiniban, panitumab, zalutuumub, nimotuzumab, mother, and mother -in -law, and nimotuzumab, and Futuzumab, blondevetmab, obinutuzumab, ibritumomab tiuxetan, tositumumab, ofatumumab, okaratuzumab, ocrelizumab, TRU-015 (trubion), veltuzumab (IMMU-106), alemtuzumab, ANT1034, HI 186(Bio Rad), YTH34.5 (Bio Rad), and YTH66.9HL ( Bio-Rad), trastuzumab and pertuzumab.

[000122] Another example of a modification that can be made to an endogenous polypeptide to obtain a truncated variant provided in this application is the removal of one or more amino acids that normally contribute to or participate in intracellular signaling and/or transport pathways. Truncation of the endogenous polypeptide to remove signaling domains enhances the function of the cellular marker as an inactive, inducible cellular marker that, when inactive, does not impair cellular functions (eg, in adopted cells during adoptive cell therapy).

[000123] In some embodiments, the cell surface polypeptide of the polypeptide construct may comprise a truncated version of the receptor tyrosine kinase. Non-limiting examples include a truncated variant of a receptor from the EGF receptor family (e.g., a truncated HER1 variant), 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 may comprise a truncated CD protein (eg, CD20 or CD52) or a truncated LNFGR (CD271).

[000124] According to certain embodiments of the present invention, the cell surface tag polypeptide may comprise a truncated variant that has been modified to remove the transmembrane domain and intracellular signaling portion of the natural or endogenous polypeptide. Truncation of the transmembrane domain and intracellular signaling frees the surface or extracellular portion of the polypeptide from its endogenous portion, e.g., making it available for insertion into a chimeric polypeptide construct that contains a cell surface polypeptide hybridized to a transmembrane domain (e.g., via a linker) derived from other natural protein. Such chimeric polypeptide constructs, in turn, can confer on a cell surface polypeptide (eg, a truncated variant) an altered activity or characteristic compared to an endogenous polypeptide variant. In some embodiments, the cell surface polypeptide is capable of dimerizing when expressed in a chimeric polypeptide construct.

[000125] The present invention provides for the expression of several different polypeptide constructs in the same modified cell. For example, a cell disclosed in this application may express several polypeptide constructs that differ in cell surface and/or transmembrane domain polypeptide identity.

[000126] The polypeptide construct described herein may comprise a cell surface polypeptide comprising a truncated version of the natural polypeptide, a transmembrane domain hybridized to the cell surface polypeptide, an optional linker connecting the truncated variant to the transmembrane domain, and a signal peptide directing a cell label to the surface of the modified cell.

Signal peptide

[000127] A signal peptide is a sequence of amino acids, typically located at the N-terminus of a newly synthesized protein or polypeptide, that directs the protein or polypeptide to the cell surface. In some embodiments of the present invention, the signal peptide directs the polypeptide to the cell surface for insertion (eg, via a transmembrane domain) into the cell membrane. In some embodiments, the polypeptide construct described herein is synthesized with a signal peptide but then post-translationally processed to cleave the signal peptide so that the mature polypeptide construct does not contain the amino acid sequence of the signal peptide. In other embodiments of the present invention, the signal peptide sequence is not cleaved and remains in the mature polypeptide construct.

[000128] The present invention provides a polypeptide construct comprising any known or unknown signal peptide capable of directing and/or transporting the polypeptide construct to the cell surface. For example, according to some embodiments of the present invention, the polypeptide construct contains a signal sequence corresponding to the signal peptide of GM-CSFRα, kappa Ig, immunoglobulin E, CD8α, TVB2 (T21A), CD52, or low affinity nerve growth factor receptor (LNGFR, TNFRSF16).

[000129] In embodiments, the signal peptide is encoded by a polynucleotide containing a nucleotide sequence that 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: 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. In some embodiments, the signal peptide contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 12; and SEQ ID NO: 14.

Peptide linker

[000130] The polypeptide construct described herein may contain a peptide linker for connecting a domain or fragment of the polypeptide construct to another domain or fragment of the polypeptide construct. In some embodiments of the present invention, a peptide linker connects the transmembrane domain of the polypeptide construct to a cell surface polypeptide (eg, a truncated natural polypeptide) of the polypeptide construct. For example, a polypeptide construct may comprise a peptide linker comprising 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 Whitlow linker.

[000131] The present invention provides a peptide linker of any length or size for connecting a transmembrane domain to a cell surface polypeptide (eg, a truncated version). For example, in some embodiments of the present invention, the size of the peptide linker is sized to maintain a distance between the truncated variant and the transmembrane domain approximately equal to the distance that exists between the native untruncated variant of the polypeptide and its endogenous transmembrane domain. In embodiments, the truncated variants described herein are connected 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 "natural" distance between HER1t and transmembrane protein. For example, if two different truncated versions of the same natural polypeptide are of different lengths, the shorter truncated version can be compensated for with a larger linker to position both truncated versions at approximately the same distance from the cell surface.

[000132] According to certain embodiments of the present invention, a peptide linker can be used to connect domains or portions thereof other than the transmembrane domain. For example, a peptide linker can connect two protein fragments of a cell surface polypeptide. In some instances, the cell surface polypeptide may be chimeric and may contain truncated variants from a variety of naturally occurring polypeptides that can be connected via a peptide linker. An example is a polypeptide construct containing HER1t together with one or more truncated variants of another member of the EGFR family (eg HER2, ErbB3 and ErbB4). In other instances, the cell surface polypeptide may comprise a concatemer of two or more copies of the truncated variant linked by a peptide linker (eg, SEQ ID NO: 123 containing two copies of the truncated CD20 polypeptide linked by an SGS linker).

[000133] In embodiments, the peptide linker is encoded by a polynucleotide containing a nucleotide sequence that 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: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; and SEQ ID NO: 23. In embodiments, the peptide linker contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a list consisting of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; and SEQ ID NO: 24.

transmembrane domain

[000134] The polypeptide construct described herein may contain a transmembrane domain that can be inserted into the plasma membrane to attach the polypeptide construct to the cell surface. The present invention provides a polypeptide construct comprising one or more of any known or unknown transmembrane domains or fragments thereof. In some embodiments, the transmembrane domain of the polypeptide construct may comprise a transmembrane domain derived from and/or homologous to one or more naturally occurring proteins. According to some embodiments of the present invention, the transmembrane domain of the polypeptide construct contains an amino acid sequence corresponding to the transmembrane domain of a single natural protein. According to some embodiments of the present invention, the transmembrane domain of the polypeptide construct comprises a chimeric transmembrane domain containing amino acid sequences derived from two or more naturally occurring proteins.

[000135] The polypeptide construct described in this application may contain a transmembrane domain that is single-pass or multi-pass. CD8α is an example of a protein having a single pass transmembrane domain. In some embodiments, the polypeptide construct disclosed herein comprises a transmembrane domain corresponding to and/or homologous to a transmembrane domain or fragment thereof from the CD8α protein. In embodiments, the transmembrane domain of the polypeptide construct is encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 33 In embodiments, the transmembrane domain comprises 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: 34.

[000136] An example of a multipass protein is CD28. In some embodiments, the transmembrane domain of the polypeptide construct may comprise a transmembrane domain corresponding to and/or homologous to the transmembrane domain or fragment thereof from the CD28 protein. In embodiments, the transmembrane domain of the polypeptide construct is encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 35 In embodiments, the transmembrane domain comprises 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: 36.

[000137] According to some embodiments of the present invention, the transmembrane domain is a dimerization transmembrane domain. As used herein, "dimerization transmembrane domain" refers to a transmembrane domain or fragment thereof that is capable of physically interacting or "dimerizing" within the plasma membrane of a cell with a second transmembrane domain or fragment thereof. Typically, the dimerization transmembrane domain is contained in the first polypeptide construct, which dimerizes at the expense of the dimerization transmembrane domain with the second polypeptide. If the transmembrane dimerization domain of the first polypeptide is hybridized at its distal (extracellular) end to the first cell surface polypeptide, and the transmembrane dimerization domain of the second polypeptide is hybridized at its distal end to the second cell surface polypeptide, the physical interaction of the first and second transmembrane domains within the cell membrane can lead to dimerization of the first and second cell surface polypeptides. According to some embodiments of the present invention, the transmembrane domain can multimerize to form a trimer, tetramer, or multimer.

[000138] According to certain embodiments of the present invention, the transmembrane dimerization domain induces dimerization of cell surface polypeptides without the need for any extracellular inducing agent (eg, a ligand or antibody specific for an epitope of the cell surface polypeptide). For example, a transmembrane dimerization domain can induce dimerization of a cell surface polypeptide by spontaneous physical interaction with or binding to a second transmembrane dimerization domain within the cell membrane of the cell. Thus, it should be understood that a cell expressing a polypeptide construct described herein may exhibit dimerized cell surface polypeptides on the cell surface prior to administration of any extracellular cell surface binding agent containing an antibody, protein, ligand, or molecule described herein. application. Dimerization of cell surface polypeptides by a transmembrane dimerization domain can increase the efficiency of a cell expressing such a polypeptide construct to enhance the cellular response when the dimerized cell surface polypeptides come into contact and recognize a ligand or antibody. For example, if the polypeptide construct contains a cell surface polypeptide containing HER1t, a pair of HER1t cell surface polypeptides can be dimerized before or during contact with a CCC or ADCC inducing agent, such as cetuximab. As a result of the dimerized configuration of cell surface polypeptides that bind cetuximab, administration of the binding agent during times of stress (eg, during a cytokine storm) can enhance the cytotoxic effect, which increases the likelihood of cell death. In some embodiments of the present invention, an agent that binds to a cell surface polypeptide (e.g., a dimerized cell surface polypeptide) to induce a cellular response in a modified cell expressing the polypeptide construct disclosed herein is provided exogenously and/or does not exist endogenously. The present invention provides a transmembrane dimerization domain to facilitate dimerization of any cell surface polypeptide, including truncated variants of natural polypeptides such as HER1t, LNGFRt, CD20t and CD52t.

[000139] According to some embodiments of the present invention, the dimerization transmembrane domain can form a covalent bond with the second transmembrane domain to induce dimerization of the cell surface polypeptide. In some embodiments of the present invention, the covalent compound is a disulfide bond formed between cysteine amino acid residues present in each of the adjacent transmembrane domains. In other embodiments, the dimerization transmembrane domain within the cell membrane can form a non-covalent bond with the second transmembrane domain to induce dimerization of the cell surface polypeptide.

[000140] The polypeptide construct described herein may contain a dimerization transmembrane domain that physically interacts with another dimerization transmembrane domain. In such cases, the first and second transmembrane dimerization domains of the respective first and second polypeptide constructs may either have the same amino acid sequence (i.e. homodimer with respect to the transmembrane dimerization domain) or different amino acid sequences (i.e. heterodimer with respect to the transmembrane domain dimerization). According to certain embodiments of the present invention, each respective transmembrane dimerization domain of a dimerized polypeptide pair contains at least one cysteine residue that mediates the formation of a disulfide bridge between the respective cysteine residues in the first and second transmembrane dimerization domains.

[000141] The transmembrane domain of the polypeptide construct may comprise an amino acid sequence corresponding to and/or homologous to the amino acid sequence of the glycophorin A protein. its fragment. In some embodiments of the present invention, the transmembrane domain of the polypeptide construct may comprise one or more amino acids that typically flank the glycophorin A transmembrane domain. ) with a second polypeptide construct containing all or part of the glycophorin A transmembrane domain. In such cases, the amino acid sequence inserted into the glycophorin A polypeptide construct may define the transmembrane dimerization domain. For example, the glycophorin A dimerization domain may contain a GXXXG dimerization motif.

[000142] In some embodiments, the polypeptide construct comprises a transmembrane domain comprising at least glycophorin A amino acids E91-R116. In some embodiments, the polypeptide construct comprises a transmembrane domain comprising at least glycophorin A amino acids 192-1114. According to embodiments, the polypeptide construct contains a transmembrane domain corresponding to a glycophorin A domain or a fragment thereof encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100 % is identical to the nucleotide sequence selected from the list consisting of SEQ ID NO: 25 and SEQ ID NO: 27. , 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 26 and SEQ ID NO: 28.

[000143] The polypeptide construct described herein may contain a transmembrane domain, which is a chimera of amino acid sequences from two or more natural polypeptides. For example, the transmembrane domain may comprise an amino acid sequence corresponding to a glycophorin A domain, or a portion thereof, fused or fused to an amino acid sequence from a second protein. According to some embodiments of the present invention, such a chimeric transmembrane domain is capable of dimerization with a second transmembrane domain (eg, chimeric or non-chimeric) and, accordingly, contains a dimerization transmembrane domain. For example, an amino acid sequence corresponding to a glycophorin A transmembrane domain or fragment thereof can be hybridized to an amino acid sequence corresponding to a β3 integrin domain or fragment thereof to form a chimeric transmembrane domain of the polypeptide construct. In some embodiments, the polypeptide construct comprises a transmembrane domain comprising glycophorin A amino acids I92-L109 hybridized to β3 integrin amino acids A737-W741. In embodiments, the polypeptide construct contains a transmembrane domain corresponding to a glycophorin A-integrin β3 chimeric sequence encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 29. In embodiments, the polypeptide construct contains a transmembrane domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical amino acid sequence of SEQ ID NO: 30.

[000144] The transmembrane domain of the polypeptide construct may comprise an amino acid sequence corresponding to and/or homologous to an amino acid sequence within the transmembrane domain of the CD3 zeta chain. In some embodiments, a polypeptide construct comprising a CD3 zeta chain transmembrane domain or fragment thereof is capable of dimerizing (e.g., homodimerizing) with a second polypeptide construct comprising a CD3 zeta chain transmembrane domain or fragment thereof. In such cases, the amino acid sequence corresponding to the CD3 zeta chain transmembrane domain may define the dimerization transmembrane domain. In some embodiments, the polypeptide construct comprises a transmembrane domain encoded by a polynucleotide comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence. SEQ ID NO: 31. According to other embodiments of the present invention, the transmembrane domain is encoded by a polynucleotide containing the nucleotide sequence of SEQ ID NO: 31. In embodiments, the polypeptide construct contains a transmembrane domain that is at least 70%, 75%, 80%, 85 %, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[000145] According to other embodiments of the present invention, the transmembrane domain of the polynucleotide construct herein may comprise an amino acid sequence corresponding to and/or homologous to the transmembrane domain or fragment thereof from CTLA4 (cytotoxic T lymphocyte protein 4) and/or LNGFR (TNFRSF16) proteins. For example, a polypeptide construct may contain a transmembrane domain encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from a list consisting of SEQ ID NO: 37 and SEQ ID NO 39. In embodiments, the polypeptide construct contains a transmembrane domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 38 and SEQ ID NO: 40.

Truncated options

[000146] The polypeptide construct described herein may comprise a cell surface polypeptide linked to a transmembrane domain. In certain embodiments of the present invention, the cell surface polypeptide comprises a truncated version of the naturally occurring polypeptide. The present invention provides a wide range of naturally occurring polypeptides as a precursor or substrate for obtaining a truncated variant incorporated into a polypeptide construct. Each natural polypeptide precursor can be further subjected to a variety of different truncations to provide a large number of possible truncated variants for use in the polypeptide constructs described herein.

[000147] Examples of precursors include epidermal growth factor receptor (EGFR or HER1) isoform a precursor (eg, SEQ ID NO: 50); an isoform a precursor of ErbB2 receptor tyrosine kinase (HER2) (eg, SEQ ID NO: 51); ErbB3 receptor tyrosine kinase (HER3) isoform 1 precursor (e.g., SEQ ID NO: 52), ErbB4 receptor tyrosine kinase (HER4) isoform JM-a/CVT-1 precursor (e.g., SEQ ID NO: 53), JM-b isoforms, isoforms X7 ErbB4 receptor tyrosine kinase (HER4) (eg, SEQ ID NO: 54), CD20 precursor (eg, SEQ ID NO: 108), CD52 precursor, and LNGFR precursor (eg, SEQ ID NO: 154).

[000148] In certain embodiments of the present invention, the cell surface polypeptide comprises a truncated HER1 polypeptide (herein referred to as HER1t or EGFRt). Natural HER1 contains an extracellular region containing domain I, II, III and IV, a transmembrane domain and an intracellular tyrosine kinase and regulatory region. Certain antibodies capable of inducing ADCC (eg, panitumumab and cetuximab) are known to bind to domain III of endogenous HER1.

[000149] The present invention provides polypeptide constructs containing HER1 polypeptides that are truncated at any amino acid, endogenous HER1 domain or fragment. According to the present invention, the HER1 polypeptide may comprise a HER1t polypeptide. In some embodiments, the HER1 polypeptide consists or essentially consists of a HER1t polypeptide. In other embodiments, the HER1 polypeptide may comprise a HER1t polypeptide in addition to other HER1 domains (eg, the HER1 transmembrane domain). In embodiments, the HER1 polypeptide may not contain an intracellular domain or fragment thereof normally found in HER1, including a tyrosine kinase domain and a regulatory region. In some embodiments, the HER1 polypeptide may not contain the transmembrane domain or fragment thereof normally found in HER1. In some embodiments, the HER1 polypeptide may not contain an extracellular domain or fragment thereof normally found in HER1, including all or part of domain I, domain II and domain IV, normally found in HER1.

[000150] In some embodiments, the cell surface polypeptide comprises a HER1t polypeptide that lacks a domain III fragment normally found in HER1. In some embodiments, the HER1t polypeptide consists or essentially consists of all or part of domain III of the endogenous HER1 protein. In some embodiments, the HER1t polypeptide consists or essentially consists of the entire domain III of the endogenous HER1 protein. In one embodiment, HER1t domain III inserted into a cell surface polypeptide is encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to nucleotide sequence of SEQ ID NO: 199. In an aqueous embodiment, the HER1t domain III inserted into the cell surface polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid the sequences of SEQ ID NO: 200.

[000151] According to some embodiments of the present invention, the HER1t polypeptide incorporated into the cell surface polypeptide comprises endogenous HER1 domain IV or a fragment thereof. The endogenous domain IV of HER1 may be encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 201. According to In one embodiment of the present invention, the HER1t endogenous 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. According to other embodiments of the present of the invention, an HER1t polypeptide inserted into a cell surface polypeptide may contain a truncated IV domain. The truncated HER1t IV domain may comprise a truncation of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% or 100% domain IV of native HER1. In one embodiment of the present invention, a truncated HER1t domain IV inserted into a cell surface polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a 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.

[000152] The polypeptide construct described in this application may contain a HER1t polypeptide containing domain III and domain IV of HER1. In one embodiment, the HER1t polypeptide comprises HER1 domain III and domain IV that are at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO : 210.

[000153] The polypeptide construct described herein may comprise an HER1t polypeptide containing HER1 domain III and a domain IV fragment of HER1. In one embodiment, the HER1t polypeptide 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: 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.

[000154] Natural HER1 also contains many pairs of disulfide bonds in domain IV. In some embodiments of the present invention, the truncated HER1t IV domain may be truncated at such positions to retain disulfide bond pairs. According to additional embodiments of the present invention, the HER1t variants described in this application are connected to the transmembrane domain through 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. According to additional embodiments of the present invention, the 7-mer linker between domain IV and the EGFR transmembrane domain can be further removed, since this linker is involved in the dimerization of EGFR receptors, which leads to activation by the EGF ligand.

[000155] A HER1t polypeptide inserted into a cell surface polypeptide described herein may contain an epitope that can be recognized by an exogenously introduced binding partner or antibody. In some embodiments, the HER1t polypeptide may contain a cetuximab-binding domain to facilitate targeted depletion of cells expressing the polypeptide construct described herein. Depletion can be caused, for example, by cell death as a result of CSC and/or ADCC. Non-limiting examples of molecules that can be introduced endogenously to bind and/or recognize an epitope on a cell surface polypeptide containing HER1t may include cetuximab, gefitinib, erlotinib, afatinib, brigatinib, icotinib, osimertinib, panitumumab, zalutumumab, nimotuzumab, and matuzumab. In various embodiments, the antibody may be a monoclonal antibody, scFv, scFab, diabody, or camelid antibody. In another embodiment, the antibody may be conjugated to a drug or toxin.

[000156] The cell surface polypeptide incorporated into the polypeptide construct described herein may contain truncated versions of many different naturally occurring polypeptides. For example, a cell surface polypeptide may comprise a polypeptide chimera containing a plurality of truncated polypeptides from the EGFR family. FIG. 4B shows an embodiment of a polypeptide construct conferring cell label functionality, wherein the cell surface polypeptide contains domain III from one HER/EGFR family member (HER(m)) and domain IV or fragment thereof from another HER/EGFR family member (HER(n )). The present invention provides chimeric cell surface polypeptides containing any combination of extracellular domains of two or more of EGFR/HER1, HER2, ErbB3 and ErbB4. FIG. 4C depicts a specific case where domain III is derived from EGFR/HER1. The advantage of such chimeric cell surface polypeptides is that each individual truncation can remove epitopes from the corresponding natural polypeptide that tend to bind endogenous molecules while maintaining an epitope that can be recognized by an exogenously introduced antibody during adoptive cell therapy. For example, if a chimeric cell surface polypeptide contains domain III from EGFR and domain IV from HER2, modified cells expressing the polypeptide may be susceptible to both cetuximab (recognizing EGFR domain III) and trastuzumab (recognizing HER2 domain IV) antibodies. Thus, the chimeric cell surface polypeptides described herein provide an additional mechanism for controlling the behavior of immune cells during immunotherapy by providing binding sites for a variety of antibiotics/binding partners. For example, in a situation where a subject experiencing side effects during adoptive cell therapy does not respond to an administered antibiotic (eg, cetuximab) that targets an epitope on one of the truncated variants in a chimeric cell surface polypeptide, another antibody may be administered to the subject ( for example, trastuzumab) that targets the same modified cell at the expense of a different epitope on a different truncated version of the chimeric cell surface polypeptide.

[000157] According to some embodiments of the present invention, the chimeric cell surface polypeptide can be hybridized to a transmembrane domain that is homologous to a member of the EGFR family. For example, the transmembrane domain may correspond to a transmembrane domain from EGFR/HER1, HER2, ErbB3, or ErbB4. According to other embodiments of the present invention, the transmembrane domain may be homologous to a non-EGFR transmembrane domain, including the transmembrane domain 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.

[000158] In some embodiments, the chimeric cell surface polypeptide may comprise a chimera of a HER1t polypeptide and a truncated HER2 (HER2t) polypeptide, or a fragment thereof. For example, the polypeptide construct may comprise a HER1t/EGFRt polypeptide containing HER1 domain III and a HER2t polypeptide containing HER2 domain IV and a HER2 transmembrane domain. According to one embodiment of the present invention, a chimeric EGFR-HER2 cell surface polypeptide hybridized to the HER2 transmembrane domain can be encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% , 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 88 and SEQ ID NO: 92 (delta 16). In one embodiment, the chimeric EGFR-HER2 cell surface polypeptide hybridized to the HER2 transmembrane domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identical. selected from the group consisting of SEQ ID NO: 89 and SEQ ID NO: 93 (delta 16).

[000159] In some embodiments, the chimeric cell surface polypeptide may comprise a chimera of an HER1t polypeptide and a truncated ErbB3 (ErbB3t) polypeptide or fragment thereof. For example, the polypeptide construct may comprise an HER1t/EGFRt polypeptide containing HER1 domain III and an ErbB3t polypeptide containing ErbB3 domain IV and an ErbB3 transmembrane domain. In one embodiment of the present invention, a chimeric EGFR-ErbB3 cell surface polypeptide hybridized to an ErbB3 transmembrane domain may be encoded by a polynucleotide comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% , 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 96. In one embodiment of the present invention, an EGFR-ErbB3 chimeric cell surface polypeptide hybridized to an ErbB3 transmembrane domain is at least 70%, 75%, 80%, 85% , 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 97.

[000160] In some embodiments, the chimeric cell surface polypeptide may comprise a chimera of an HER1t polypeptide and a truncated ErbB4 (ErbB4t) polypeptide or fragment thereof. For example, the polypeptide construct may comprise a HER1t/EGFRt polypeptide containing domain III and an ErbB4t polypeptide containing ErbB4 domain IV and an ErbB4 transmembrane domain. In some embodiments of the present invention, the ErbB4t polypeptide may comprise a JM-a extracellular perimembrane domain encoded by an alternative ErbB4 JM-a transcript. In some embodiments, the ErbB4t polypeptide may comprise a JM-b extracellular perimembrane domain encoded by an alternative ErbB4 JM-b transcript. In one embodiment of the present invention, the chimeric cell surface polypeptide hybridized to the ErbB4 transmembrane domain can be encoded by a polynucleotide containing a nucleotide sequence that 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: 100 (EGFR-ErbB4 (JM-a)) and SEQ ID NO: 104 (EGFR-ErbB4 (JM-b)). In one embodiment, the chimeric EGFR-ErbB4 cell surface polypeptide hybridized to the ErbB4 transmembrane domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identical. selected from the list consisting of SEQ ID NO: 101 (EGFR-ErbB4 (JM-a)) and SEQ ID NO: 105 (EGFR-ErbB4 (JM-b)).

[000161] The polypeptide construct described herein may contain any combination of a signal peptide, a cell surface polypeptide containing a HER1t polypeptide (for example, containing only HER1t or a chimeric polypeptide containing HER1t), a transmembrane domain, and an optional linker. For example, the polypeptide construct may comprise a cell surface polypeptide comprising a HER1t polypeptide or a chimeric HER1t containing polypeptide fused or hybridized to a CD28 transmembrane domain derivative or fragment via a linker (e.g., one or more (e.g., 1-4) copies of (G4S (SEQ ID NO: 221))). In some embodiments, the cell surface polypeptide may comprise a HER1t polypeptide encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical 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). In some embodiments, the cell surface polypeptide may comprise a HER1t polypeptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid 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 (HER1t3); 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).

[000162] According to some embodiments of the present invention, the polypeptide construct may comprise a signal peptide, a cell surface polypeptide containing a HER1t polypeptide, or a chimeric polypeptide containing a HER1t polypeptide, a transmembrane domain containing a dimerization transmembrane domain, and a linker for connecting the transmembrane domain and the cell surface polypeptide . For example, a polypeptide construct may comprise a kappa Ig signal peptide, a specific HER1 truncated variant, a linker (e.g., (G4S)4 (SEQ ID NO: 22)) and a dimerization transmembrane domain (e.g., containing I92-I114 of glycophorin A or a transmembrane domain from CD3 -zeta). In embodiments, a polypeptide construct comprising a HER1t polypeptide and a transmembrane dimerization domain is encoded by a polynucleotide containing a nucleotide sequence that 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: 70 (glycophorin A; HER1t8); SEQ ID NO: 74 (glycophorin A; HER1t9); SEQ ID NO: 78 (glycophorin A; HER1t10); and SEQ ID NO: 82 (CD3-zeta; HER1t11). In embodiments, the polypeptide construct comprising the HER1t polypeptide and the dimerization transmembrane domain contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence, selected from the list consisting of SEQ ID NO: 71 (glycophorin A; HER1t8); SEQ ID NO: 75 (glycophorin A; HER1t9); SEQ ID NO: 79 (glycophorin A; HER1t10); and SEQ ID NO: 83 (CD3-zeta; HER1t11).

[000163] In another embodiment, the polypeptide construct may comprise a cell surface polypeptide comprising the HER1t-HER2t chimera fused to the HER2 transmembrane domain. The truncated HER1t-HER2t chimera and transmembrane domain can be further linked to a signal peptide (eg, GM-CSFRα) that directs the polypeptide construct to the cell surface. According to one embodiment of the present invention, the polypeptide construct is encoded by a polynucleotide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from a list consisting of SEQ ID NO: 86 and SEQ ID NO: 90 (delta 16). According to one embodiment of the present invention, the polypeptide construct has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a list consisting of SEQ ID NO: 87 and SEQ ID NO: 91 (delta 16).

[000164] In another example, the polypeptide construct may comprise a cell surface polypeptide containing the HER1t-ErbB3t chimera fused to the transmembrane domain of ErbB3. In one embodiment of the present invention, the HER1t-ErbB3t chimera and the transmembrane domain are further linked to a signal peptide (eg, GM-CSFRα) that directs the polypeptide construct to the cell surface. According to one embodiment of the present invention, the polypeptide construct is encoded by a polynucleotide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 94. According to one In an embodiment of the present invention, the polypeptide 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.

[000165] In yet another example, the polypeptide construct may comprise a cell surface polypeptide containing the HER1t-ErbB4t chimera fused to the ErbB4 transmembrane domain. In one embodiment of the present invention, the HER1t-ErbB4t chimera and the transmembrane domain are further linked to a signal peptide (eg, GM-CSFRα) that directs the polypeptide construct to the cell surface. According to one embodiment of the present invention, the polypeptide construct is encoded by a polynucleotide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from a list consisting of SEQ ID NO: 98 (variant JM-a) and SEQ ID NO: 102 (variant JM-b). According to one embodiment of the present invention, the polypeptide construct has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a list consisting of SEQ ID NO: 99 (version JM-a) and SEQ ID NO: 103 (version JM-b).

[000166] The polypeptide construct containing the HER1t polypeptide may contain any signal peptide capable of directing the polypeptide construct to the cell surface. For example, a cell surface polypeptide containing a HER1t polypeptide (e.g., containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a 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 hybridized to a signal peptide containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GM-CSFRα), SEQ ID NO: 4 (kappa Ig), 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).

[000167] A polypeptide construct containing a HER1t polypeptide may contain any transmembrane domain, including a transmembrane domain that does not contain a dimerization domain. For example, a cell surface polypeptide containing a HER1t polypeptide (e.g., containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a 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 linked to a transmembrane domain containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical 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), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTLA4) and SEQ ID NO: 40 (LNGFR).

[000168] A polypeptide construct containing a HER1t polypeptide may contain any peptide linker (or, in some embodiments, no peptide linker). For example, a cell surface polypeptide containing HER1t (for example, containing an amino acid sequence that 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: 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 hybridized to a peptide linker containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the 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).

[000169] The cell surface polypeptide may comprise a truncated CD polypeptide. For example, the cell surface polypeptide may contain a truncated CD20 polypeptide (referred to in this application as CD20t). The native CD20 polypeptide is a multipass transmembrane protein encoded by gene 1 of subfamily A of the family of genes encoding proteins with 4 transmembrane domains (MS4A1). According to certain embodiments of the present invention, full-length CD20 may be encoded by a polynucleotide containing the nucleotide sequence of SEQ ID NO: 106, and the full-length amino acid sequence of CD20 may correspond to the amino acid sequence of SEQ ID NO: 107. In some embodiments of the present invention, CD20 contains 4 passages of the transmembrane domain containing amino acids 57-78, 85-105, 121-141 and 189-209. According to some embodiments of the present invention, CD20 contains 2 extracellular domains containing amino acids 79-84 and 142-188. According to some embodiments of the present invention, CD20 contains 3 cytoplasmic domains containing amino acids 1-56, 106-120 and 210-297.

[000170] The present invention provides polypeptide constructs containing CD20 polypeptides that are truncated at any amino acid, endogenous CD20 domain or fragment. According to the present invention, a CD20 polypeptide may comprise a CD20t polypeptide. In some embodiments, the CD20 polypeptide consists of, or essentially consists of, a CD20t polypeptide. In other embodiments, the CD20 polypeptide may comprise a CD20t polypeptide in addition to another CD20 domain or portion thereof (eg, a CD20 transmembrane domain and/or a cytoplasmic domain). For example, a CD20 polypeptide may be truncated at an intracellular cytoplasmic (eg, signaling) domain or portion thereof, a transmembrane (eg, helical) domain or portion thereof, and/or an extracellular domain or portion thereof. In some embodiments of the present invention, a CD20 polypeptide may lack several domains or several portions of a domain relative to the wild-type polypeptide. In one embodiment, the CD20 polypeptide comprises M1-E263 of endogenous CD20 (SEQ ID NO: 109; CD20I1), M117-N214 of endogenous CD20 (SEQ ID NO: 111 (CD20t2), M1-N214 of endogenous CD20 (SEQ ID NO: 115 ; CD20t4), V82-N214 endogenous CD20 (SEQ ID NO: 117; CD20t5) or V82-I186 endogenous CD20 (SEQ ID NO: 119, CD20t6).

[000171] According to one implementation variant of the present invention, the CD20t polypeptide may contain an extracellular domain or a fragment thereof. In one embodiment, the CD20t polypeptide 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: 218 , SEQ ID NO: 219 and SEQ ID NO: 220. In some embodiments of the present invention, the CD20t polypeptide may be linked to a transmembrane domain or fragment thereof. According to one implementation variant of the present invention, the polypeptide construct contains a CD20t polypeptide connected to the transmembrane domain of CD20. In some embodiments of the present invention, a polypeptide construct comprising a CD20t polypeptide fused to a CD20 transmembrane domain is encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 % or 100% identical to the nucleotide sequence selected from the list consisting of SEQ ID NO: 108 (CD20t1 encoding M1-E263); SEQ ID NO: 110 (CD20t2 encoding M117-N214); SEQ ID NO: 114 (CD20t4 encoding M1-N214); SEQ ID NO: 116 (CD20t5 encoding V82-N214); SEQ ID NO: 118 (CD20t6 encoding V82-I186); SEQ ID NO: 132 (CD20t13 encoding M1-A54 IS111-P297); SEQ ID NO: 134 (CD20t14 encoding M1-A54 and C111-E281); SEQ ID NO: 136 (CD20t15 encoding M1-A54 and C111-E263); SEQ ID NO: 138 (CD20t16 encoding M1-A54 and C111-V228); and SEQ ID NO: 140 (CD20t17 encoding M1-V8 and C111-P297). According to one embodiment of the present invention, a polypeptide construct comprising a CD20t polypeptide fused to a CD20 transmembrane domain may contain an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 109 (CD20t1: M1-E263); SEQ ID NO: 111 (CD20t2: M117-N214); SEQ ID NO: 115 (CD20t4: M1-N214); SEQ ID NO: 117 (CD20t5: V82-N214); SEQ ID NO: 119 (CD20t6: V82-I186); SEQ ID NO: 133 (CD20t13: M1-A54 and C111-P297); SEQ ID NO: 135 (CD20t14: M1-A54 and C111-E281); SEQ ID NO: 137 (CD20t15: M1-A54 and C111-E263); SEQ ID NO: 139 (CD20t16: M1-A54 and C111-V228); and SEQ ID NO: 141 (CD20t17: M1-V8 and C111-P297).

[000172] In some embodiments, the CD20t polypeptide can retain at least one copy of the endogenous CD20 rituximab-binding mimotope described in Philip et al., (2014), “A highly compact epitope-based marker/suicide gene for easier and safer T-cell tHERapy," Blood: 124: 1277-1287. In some embodiments, one or more copies of the rituximab-binding CD20t epitope can be fused to one or more amino acid sequences derived from CD-34, such as the 40 amino-terminal amino acids of CD34, which facilitate binding of the anti-CD34 monoclonal antibody. In some embodiments of the present invention, several domains may be removed from a CD20t polypeptide, or a portion of several domains may be removed.

[000173] A cell surface polypeptide containing a CD20t polypeptide may contain an epitope that can be recognized by an exogenously introduced antibody or binding partner. For example, a CD20t polypeptide incorporated into a cell surface polypeptide may contain a rituximab binding domain to facilitate targeted depletion of cells expressing the polypeptide construct described herein (e.g., by CSC and/or antibody-dependent cellular cytotoxicity (ADCC)). Non-limiting examples of antibodies that can be used to bind to an epitope of a cell surface polypeptide containing a CD20t polypeptide include rituximab, hOUBM3/6, afutuzumab, blontuvetmab, obinutuzumab, ibritumomab tiuxetan, tositumumab, ofatumumab, ocaratuzumab, ocrelizumab, TRU-015 (trubion), and veltuzumab (IMMU-106). In various embodiments, the antibody may be a monoclonal, scFv, scFab, diabody, or camelid antibody. In other embodiments of the present invention, the antibody may be conjugated to a drug or toxin. In some embodiments, the CD20t polypeptide contains an epitope found in the extracellular domain of endogenous CD20. In some embodiments, the CD20t polypeptide epitope contains amino acids 170-185 of endogenous CD20. In some embodiments, the CD20t polypeptide epitope comprises amino acids 170-173 and 182-185 of endogenous CD20.

[000174] According to further embodiments of the present invention, a cell surface polypeptide comprising a CD20t polypeptide is chimeric to a truncated version of one or more additional polypeptides. For example, a chimeric cell surface polypeptide may comprise a CD20t polypeptide and a truncated CD8α (CD8αt) polypeptide.

[000175] The cell surface polypeptide may further comprise several CD20t sequences linked together as a concatemer. For example, a cell surface polypeptide may contain the same CD20t amino acid sequence repeated consecutively, or different CD20t variants joined together. In some embodiments of the present invention, the CD20t amino acid sequences are linked together in a cell surface polypeptide using a peptide linker. In one embodiment of the present invention, an SGS or SG4S linker (SEQ ID NO: 223) can be used to link together repetitive CD20 amino acid sequences in a cell surface polypeptide (e.g., see SEQ ID NO: 123 (SGS linker) and SEQ ID NO: 129 (SG4S linker (SEQ ID NO: 223)), which further comprises a cell surface polypeptide linked to the CD28 transmembrane domain via an SG4S linker (SEQ ID NO: 223).

[000176] The polypeptide construct described herein may contain any combination of a signal peptide, a cell surface polypeptide containing a CD20t polypeptide (for example, containing only CD20t or a CD20t chimera), a transmembrane domain, and an optional linker. For example, the polypeptide construct may contain an SG4S linker (SEQ ID NO: 223) that connects or hybridizes a cell surface polypeptide containing a CD20t polypeptide to a transmembrane domain. In certain embodiments of the present invention, the transmembrane domain is derived from or homologous to the transmembrane domain or fragment thereof of CD20, CD28, or CD8α. According to one embodiment of the present invention, a CD20t-containing polypeptide construct is encoded by a polynucleotide comprising a nucleotide sequence selected from the list consisting of SEQ ID NO: 112 (CD20t3 encoding CD20t (K142-S188) and the CD8α transmembrane domain (I183-T203) ); SEQ ID NO: 120 (CD20t7 encoding CD20t (P160-Q187), SG4S linker (SEQ ID NO: 223) and CD28 transmembrane domain (I96-D172)); SEQ ID NO: 122 (CD20t8 encoding CD20t concatemer (P160-Q187 separated by SGS linker), SG4S linker (SEQ ID NO: 223) and CD28 transmembrane domain (I96-D172)); SEQ ID NO: 124 (CD20t9 encoding CD20t (P160-Q187), SG4S linker (SEQ ID NO: 223) and CD8α transmembrane domain (P120-V201)); SEQ ID NO: 126 (CD20t10 encoding CD20t (C167-C183), SG4S linker (SEQ ID NO: 223) and CD28 transmembrane domain (I96-D172); SEQ ID NO: 128 (CD20t11 encoding CD20 concatemer (C167-C183 separated by an SG4S linker (SEQ ID NO: 223)), an SG4S linker (SEQ ID NO: 223), and a CD28 transmembrane domain (I96-D172); and SEQ ID NO: 130 (CD20t12 encoding CD20t (C167-C183), a SG4S (SEQ ID NO: 223) and CD8α transmembrane domain (P120-V201)).In one embodiment of the present invention, the polypeptide construct comprising CD20t comprises an amino acid sequence selected from the list consisting of SEQ ID NO: 113 (CD20t3; CD20t (K142-S188) and CD8α transmembrane domain (I183-T203)); SEQ ID NO: 121 (CD20t7 encoding CD20t (P160-Q187), SG4S linker (SEQ ID NO: 223) and CD28 transmembrane domain (I96-D172) ); SEQ ID NO: 123 (CD20t8 encoding CD20t concatemer (P160-Q187 separated by SGS linker), SG4S linker (SEQ ID NO: 223) and CD28 transmembrane domain (I96-D172)); SEQ ID NO: 125 (CD20t9 encoding CD20t (P160-Q187), SG4S linker (SEQ ID NO: 223) and CD8α transmembrane domain (P120-V201)); SEQ ID NO: 127 (CD20t10 encoding CD20t (C167-C183), SG4S linker (SEQ ID NO: 223) and CD28 transmembrane domain (I96-D172); SEQ ID NO: 129 (CD20t11 encoding CD20 concatemer (C167-C183 separated by an SG4S linker (SEQ ID NO: 223)), an SG4S linker (SEQ ID NO: 223), and a CD28 transmembrane domain (I96-D172); and SEQ ID NO: 131 (CD20t12 encoding CD20t (C167-C183), linker SG4S (SEQ ID NO: 223) and CD8α transmembrane domain (P120-V201)).

[000177] A polypeptide construct containing a CD20t polypeptide may contain any signal peptide capable of directing the polypeptide construct to the cell surface. For example, a polypeptide construct containing a CD20t polypeptide (for example, a polypeptide that contains an amino acid sequence selected from the list consisting of SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220) can be hybridized to a signal peptide, containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GM- CSFRα), SEQ ID NO: 4 (kappa Ig), 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).

[000178] A polypeptide construct containing a CD20t polypeptide may contain any transmembrane domain, including a transmembrane domain that contains a dimerization domain or does not contain a dimerization domain. For example, a polypeptide construct containing a CD20t polypeptide (for example, a polypeptide that contains an amino acid sequence selected from the list consisting of SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220) can be hybridized to a transmembrane domain, containing an amino acid sequence that 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: 26 (glycophorin A E91-R116), SEQ ID NO: 28 (glycophorin A 192-1114), SEQ ID NO: 30 (glycophorin A (I92-L109) β3 integrin (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). derived from or homologous to CD28 and/or CD8α.

[000179] A polypeptide construct comprising a CD20t polypeptide may contain any peptide linker (or, in some embodiments, no peptide linker). For example, a polypeptide construct containing a CD20t polypeptide (for example, a polypeptide that contains an amino acid sequence selected from the list consisting of SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220) can be hybridized to a peptide linker, containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the 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).

[000180] Another example of a CD polypeptide that can be truncated and inserted into a cell tag as described herein is CD52. CD52 occurs endogenously in humans as a 12 amino acid peptide linked at its C-terminus to a glycosylphosphatidylinositol (GPI) anchor. In some embodiments of the present invention, glycophosphatidylinositol (GPI) can be used to attach the polypeptides described herein to a cell surface.

[000181] The present invention provides polypeptide constructs containing CD52 polypeptides that are truncated at any amino acid, endogenous CD52 domain or fragment. According to the present invention, a CD52 polypeptide may comprise a truncated CD52 (CD52t) polypeptide. In some embodiments, the CD52 polypeptide consists or essentially consists of a cell surface polypeptide comprising a CD52t polypeptide. In other embodiments, the CD52 polypeptide may comprise a cell surface polypeptide comprising a CD52t polypeptide in addition to other CD52 domains (eg, a CD52 signal peptide).

[000182] The present invention provides polypeptide constructs that comprise a cell surface polypeptide containing a truncated CD52t polypeptide. In some embodiments of the present invention, a CD52t polypeptide is coupled to a CD52 signal peptide to target the truncated variant to the cell surface. In some embodiments, a cell surface polypeptide comprising a CD52t polypeptide may contain one or more epitopes that can be recognized by an exogenously introduced antibody or binding partner. For example, a CD52t polypeptide may contain one or more alemtuzumab binding domains and thereby facilitate targeted depletion of cells expressing the polypeptide construct described herein. In some embodiments of the present invention, targeted depletion may result from alemtuzumab-mediated CCC and/or ADCC, or another cellular mechanism that mediates the cytotoxic effects of alemtuzumab recognition. Non-limiting examples of anti-CD52 molecules that can recognize a cell surface polypeptide containing a CD52t polypeptide include alemtuzumab, ANT1034, HI 186 (Bio Rad), YTH34.5 (Bio Rad) and YTH66.9HL (Bio-Rad). According to various embodiments of the present invention, the antibody may be a monoclonal antibody, scFv, scFab, diabody, or camelid antibody. According to one embodiment of the present invention, the polynucleotide sequence encoding the CD52 epitope contains a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 142. According to one embodiment of the present invention, the CD52 epitope is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 143.

[000183] The polypeptide construct described herein may have multiple epitopes that can be recognized by an antibody or binding partner. For example, in the case of a CD52t polypeptide, a polypeptide construct containing a CD52t polypeptide may contain multiple epitopes specific for a binding partner (eg, alemtuzumab). In one embodiment, the CD52t polypeptide contains multiple copies (e.g., at least two copies, at least three copies, at least four copies, at least five copies, at least six copies, or at least ten copies) the amino acid sequence shown in SEQ ID NO: 143. Each copy or repeat of the amino acid sequence can be separated in the polypeptide construct by a linker (eg, a Whitlow linker). In one embodiment of the present invention, the polypeptide construct comprises a CD52 signal peptide linked to one or more copies of a CD52t polypeptide hybridized to a Whitlow linker, which is linked (e.g., via one or more copies of a (G4S) linker (SEQ ID NO: 221)) with a transmembrane domain (eg, CD28 transmembrane domain or fragment thereof). In one embodiment of the present invention, a polypeptide construct comprising a CD52t polypeptide is encoded by a polynucleotide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a nucleotide sequence selected from a list consisting of SEQ ID NO: 144 (CD52t1; encoding one copy of the epitope/linker), SEQ ID NO: 146 (CD52t2; encoding two copies of the epitope/linker), and SEQ ID NO: 148 (CD52t3; encoding three copies of the epitope/linker ). In one embodiment of the present invention, a polypeptide comprising a CD52t polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from a list consisting of SEQ ID NO: 145 (CD52t1; encoding one copy of the epitope/linker;), SEQ ID NO: 147 (CD52t2; encoding two copies of the epitope/linker), and SEQ ID NO: 149 (CD52t3; encoding three copies of the epitope/linker).

[000184] A polypeptide construct containing a CD52t polypeptide may contain any transmembrane domain, including a transmembrane domain that contains a dimerization domain or does not contain a dimerization domain. For example, a cell surface polypeptide containing a CD52t polypeptide can be hybridized via a linker (e.g., a 3xGS linker (SEQ ID NO: 224)) to a transmembrane domain containing a dimerization transmembrane domain (e.g., derived from the transmembrane domain of glycophorin A or glycophorin A- integrin β3 or homologous to them). In one embodiment of the present invention, a polypeptide construct comprising a CD52t polypeptide 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: 150 (CD52t4; CD52 signal peptide, 3xGS peptide linker (SEQ ID NO: 224) and glycophorin A transmembrane domain), SEQ ID NO: 151 (CD52t5; CD52 signal peptide, 3xGS peptide linker (SEQ ID NO : 224) and glycophorin A transmembrane domain) and SEQ ID NO: 152 (CD52t6; CD52 signal peptide, 3×GS linker (SEQ ID NO: 224) and glycophorin A-integrin β3 transmembrane domain).

[000185] In embodiments, a polypeptide construct comprising a CD52t polypeptide (e.g., SEQ ID NO: 143) that can be fused to a CD52 signal peptide to produce an amino acid sequence that 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: 145, SEQ ID NO: 147, and SEQ ID NO: 149), can be linked to a transmembrane domain, containing an amino acid sequence that 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: 26 (glycophorin A E91-R116), SEQ ID NO: 28 (glycophorin A I92-I114), SEQ ID NO: 30 (glycophorin A(I92-L109).β3 integrin (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).

[000186] A polypeptide construct containing a CD52t polypeptide may contain any signal peptide capable of directing the polypeptide construct to the cell surface. In one embodiment of the present invention, a CD52t polypeptide is fused to a CD52 signal peptide (e.g., the polypeptide construct contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the 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 (e.g., SEQ ID NO: 143) coupled to a signal peptide containing an amino acid sequence that 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: 2 (GM-CSFRα), SEQ ID NO: 4 (kappa Ig), 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).

[000187] A polypeptide construct comprising a CD52t polypeptide may contain any peptide linker (or, in some embodiments, no peptide linker). For example, a cell surface polypeptide containing CD52t (for example, containing 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: 143) can be hybridized with a peptide linker containing an amino acid sequence that 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: 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).

[000188] According to other embodiments of the present invention, the cell surface polypeptide may comprise a truncated version of a polypeptide from the tumor necrosis factor (TNF) receptor superfamily. For example, LNGFR is a type I single-pass transmembrane glycoprotein having an extracellular domain with a folded structure due in part to the formation of a disulfide bond between cysteine residues in the protein. According to one embodiment of the present invention, a polynucleotide encoding an LNGFR, or a portion thereof, contains a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence, selected from the list consisting of SEQ ID NO: 153, SEQ ID NO: 155 (coding for K29-N250 of the LNGFR extracellular domain), SEQ ID NO: 157 (coding for E65-N250, including cysteine residues 2, 3, 4 capable of forming disulfide bonds) and SEQ ID NO: 159 (encoding R108-N250 including cysteine residues 3, 4 capable of forming disulfide bonds). According to one embodiment of the present invention, the LNGFR 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: 154, SEQ ID NO: 156 (containing K29-N250 of the LNGFR extracellular domain), SEQ ID NO: 158 (containing E65-N250, including cysteine residues 2, 3, 4 capable of forming disulfide bonds) and SEQ ID NO: 160 (containing R108- N250, including cysteine residues 3, 4 capable of forming disulfide bonds).

[000189] The present invention provides polypeptide constructs containing LNGFR polypeptides that are truncated at any amino acid, endogenous LNGFR domain or fragment. According to the present invention, the LNGFR polypeptide may comprise an LNGFRt polypeptide. In some embodiments, the LNGFR polypeptide consists or essentially consists of a cell surface polypeptide comprising an LNGFRt polypeptide. In other embodiments, the LNGFR polypeptide may comprise a cell surface polypeptide comprising an LNGFRt polypeptide in addition to other LNGFR domains (eg, the LNGFR transmembrane domain).

[000190] The polypeptide construct may comprise a truncated LNGFR polypeptide (here LNGFRt), with any domain or fragment thereof truncated relative to the wild-type protein. For example, the LNGFRt polypeptide may be truncated at one or more of the transmembrane domain or portion thereof, the intracellular domain or portion thereof, or the extracellular domain or portion thereof. In some embodiments, the LNGFRt polypeptide may be truncated at one or more TNFR-Cys repeats that form a natural binding domain for its ligands (eg, NGF, BDNF, NTF3, and NTF4). According to one implementation variant of the present invention, the polypeptide construct contains a LNGFRt polypeptide containing the entire extracellular domain (SEQ ID NO: 156). In one embodiment of the present invention, a polypeptide construct comprising an LNGFRt polypeptide is hybridized to the transmembrane domain of LNGFR. In one embodiment of the present invention, a polynucleotide encoding an LNGFRt polypeptide fused to an LNGFR transmembrane domain contains a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 161 (LNGFRt1). In one embodiment of the present invention, the LNGFRt polypeptide linked to the LNGFR transmembrane domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 162 (LNGFRt1).

[000191] The polypeptide construct containing the LNGFRt polypeptide may contain any transmembrane domain, including a transmembrane domain that contains a dimerization domain or does not contain a dimerization domain. For example, the polypeptide construct may comprise an LNGFRt polypeptide fused to an LNGFR transmembrane domain or fragment thereof. In other embodiments, the LNGFRt polypeptide may be hybridized (eg, via a linker) to a transmembrane domain or fragment thereof derived from another polypeptide, including a dimerizable transmembrane domain. In one embodiment of the present invention, a polypeptide construct comprising an LNGFRt polypeptide 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: 163 (LNGFRt2; full extracellular domain of LNGFR; GS linker and transmembrane domain of CD28); SEQ ID NO: 164 (LNGFRt3; LNGFR extracellular domain fragment including cysteine residues 2-4, GS peptide linker and CD28 transmembrane domain), SEQ ID NO: 165 (LNGFRt3; LNGFR extracellular domain fragment including cysteine residues 3-4, peptidic GS linker and CD28 transmembrane domain); SEQ ID NO: 166 (LNGFRt5; LNGFR extracellular domain fragment including cysteine residues 3-4, GS linker and glycophorin A transmembrane domain), SEQ ID NO: 167 (LNGFRt6; LNGFR extracellular domain fragment including cysteine residues 3-4, linker GS and the transmembrane domain of glycophorin A); and SEQ ID NO: 168 (LNGFRt7; LNGFR extracellular domain fragment containing cysteine residues 3-4, GS linker, and glycophorin A-integrin β3 transmembrane domain).

[000192] According to some embodiments of the present invention, a polypeptide construct comprising an LNGFRt polypeptide can be hybridized to a transmembrane domain containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the 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). β3 integrin (A737-W741), SEQ ID NO: 32 (CD3 zeta), SEQ ID NO: 34 (CD8α), SEQ ID NO: 36 (CD28), SEQ ID NO: 38 (CTLA4 ) and SEQ ID NO: 40 (LNGFR).

[000193] The polypeptide construct containing the LNGFRt polypeptide may contain any signal peptide capable of directing the polypeptide construct to the cell surface. For example, a cell surface polypeptide containing an LNGFRt polypeptide can be hybridized to a signal peptide containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical. amino acid sequence selected from the list consisting of SEQ ID NO: 2 (GM-CSFRα), SEQ ID NO: 4 (kappa Ig), 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).

[000194] A polypeptide construct containing an LNGFRt polypeptide may contain any peptide linker (or, in some embodiments, no peptide linker). For example, a cell surface polypeptide containing an LNGFRt polypeptide can be hybridized to a peptide linker containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical 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).

Polynucleotide constructs

Vector

[000195] The polypeptide constructs described herein may be encoded by one or more polynucleotides inserted into the modified cell via a vector. As used herein, an "expression vector" or "vector" is any genetic element, e.g., plasmid, chromosome, virus, transposon, that functions as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replication under its own control) or becomes capable of replication by insertion into the chromosome of the host cell by attaching another polynucleotide segment thereto to effect replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and cosmids. Vectors may contain polynucleotide sequences that are necessary to effect ligation or insertion of the vector into the target host cell and to effect expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences for transcription, enhancer sequences for enhancing transcription, ribosome binding site sequences, and transcription and translation termination sequences. In another embodiment, expression vectors may be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences.

[000196] The vector may also contain a "selectable marker gene". As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows specific positive or negative selection of cells expressing the nucleic acid sequence in the presence of an appropriate selective agent. Suitable selectable marker genes are known in the art and are described, for example, in international patent application publications WO 1992/08796 and WO 1994/28143; 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 (1987); Wigler et al. Cell 11:223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. sci. USA 48: 2026 (1962); Lowy et al. Cell 22:817 (1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[000197] According to some embodiments of the present invention, the vector is an "episomal expression vector" or "episome" that is capable of replicating in a host cell and maintained as an extrachromosomal segment of DNA in the host cell in the presence of an appropriate selective pressure (see, for example, Conese et al., Gene THERapy, 11:1735-1742 (2004)). Exemplary commercially available episomal expression vectors include, but are not limited to, episomal plasmids utilizing the Epstein-Barr virus (EBNA1) core antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The pREP4, pCEP4, pREP7 and pcDNA3.1 vectors from Invitrogen (Carlsbad, CA, USA) and pBK-CMV from Stratagene (La Jolla, CA, USA) are non-limiting examples of an episomal vector that uses a T antigen and an origin replication of SV40 instead of EBNA1 and oriP.

[000198] The present invention provides a polypeptide construct that may comprise a signal peptide, a transmembrane domain, a cell surface polypeptide containing a truncated version of the natural polypeptide, and an optional peptide linker connecting the transmembrane domain to the cell surface polypeptide. For example, in FIG. 4A illustrates an embodiment of a polypeptide construct having the function of a cell label. The truncated variant protrudes into the extracellular matrix via a peptide linker that binds to the transmembrane domain. The truncated variant may contain one or more epitopes for an antibody directed against the truncated variant (eg, added exogenously) that can recognize and bind to the truncated variant/cell label. In some embodiments of the present invention, binding of an antibody to a truncated variant may result in cell depletion (e.g., through ADCC), which may be beneficial, for example, during adoptive cell therapy where the cell has been modified to express one or more additional molecules, such as as a cytokine, TCR and/or CAR. In some embodiments of the present invention, different polypeptide constructs contain a constant transmembrane domain and a signal peptide, but differ in the identity of the truncated variant inserted into the polypeptide construct. For example, different polypeptide constructs may contain different truncated versions of the same natural polypeptide. If two polypeptide constructs contain different truncated versions of the same natural polypeptide, the polypeptide constructs may further differ depending on the presence/absence of a peptide linker in the polypeptide construct or the length of the peptide linker. In some embodiments of the present invention, 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.

[000199] The present invention provides polynucleotides and methods for using polynucleotides to facilitate the construction of polypeptide constructs for use during immunotherapy. In certain embodiments of the present invention, the polynucleotide may comprise a vector containing a sequence encoding a signal peptide and a transmembrane domain (eg, either containing or not containing a dimerization transmembrane domain). The vector may provide for cloning of an insert between the signal peptide and the transmembrane domain such that the insert contains a nucleotide sequence encoding a particular truncated variant and optionally a peptide linker. In another embodiment, vectors can be provided that contain a polynucleotide encoding a signal peptide, a specific truncated variant, and optionally a linker, and insert a nucleotide sequence encoding a transmembrane domain adjacent to the coding sequence for the truncated variant or linker. The present invention facilitates the production of polypeptide constructs containing any combination of signal peptide, truncated variant, transmembrane domain, and linker by providing a range of vectors that retain a particular domain or domains as constant in the final polypeptide construct, but allow a domain exchange that can vary between polypeptide constructs.

Vector modifications

[000200] A polynucleotide vector suitable for the methods and compositions described herein may be a Good Manufacturing Practice (GMP) vector. For example, a GMP vector may be cleaner than a non-GMP vector. In some cases, purity can be measured using bioburden. For example, the bioburden may be the presence or absence of aerobes, anaerobes, sporeformers, fungi, or combinations thereof in the vector composition. In some cases, a pure vector may be low in endotoxin or endotoxin free. Purity can also be measured by double strand sequencing using primer-walking. The identity of the plasmid can be a source of determining the purity of the vector. A GMP vector according to the present invention can be 10-99% purer than a non-GMP vector. GMP vector 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 the non-GMP vector, as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof.

[000201] In some cases, a terminator sequence is applied at the end of the first gene program. The terminator sequence can ensure that transcript synthesis is completed before the start of the second gene program. For example, expression vectors may contain sequences necessary for terminating transcription and for stabilizing the mRNA. Such sequences are usually accessible from the 5', and sometimes from the 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions may contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA. Cells containing the expression vector are grown under conditions that allow expression of the target polypeptide, either in vivo or in vitro.

[000202] In some cases, a spacer sequence may be used at the end of the first polypeptide encoded by the polynucleotide in the vector. In other cases, a spacer sequence may be used at the end of the second gene in the vector. The spacer sequence can also be applied after the first gene and the second gene in the vector.

[000203] These vectors can be used to express a polypeptide encoded by a gene or part of a gene of interest. The gene portion or gene may be inserted using any method, whether viral or non-viral. For example, the method may be a non-viral technique.

Additional features encoded by constructs

[000204] The polynucleotides disclosed herein may encode one or more proteins in addition to or simultaneously with the polynucleotide constructs described above. For example, in some embodiments of the present invention, the polypeptide constructs can be coupled to a chimeric antigen receptor (CAR), a T cell receptor (TCR), and/or a cytokine.

Chimeric receptors

[000205] Some embodiments described herein include a polynucleotide that encodes a chimeric receptor expressed on a cell surface. In some cases, the chimeric receptor contains an antigen-binding region that allows recognition and binding to an antigen, for example, a tumor antigen, such as a tumor-associated antigen or a tumor-specific antigen. In some cases, the antigen-binding region contains an antibody or its binding fragment, for example, Fab, Fab', F(ab') ₂ , F(ab') ₃ , scFv, sc(Fv)2, dsFv, diabody, minibody and nanobody or their linking fragments. In some cases, the antigen-binding region contains scFv. In some cases, the chimeric receptor contains scFv (eg, chimeric antigen receptor (CAR)). In some cases, the chimeric antigen receptor contains a pattern recognition receptor. In other cases, the chimeric receptor contains a modified T cell receptor (TCR).

Chimeric antigen receptors (CAR)

[000206] In some embodiments, the cell expressing the polypeptide construct described herein also expresses one or more chimeric antigen receptors (CARs).

[000207] The chimeric antigen receptor (CAR) described herein is a modified receptor that confers exogenous specificity on an immune effector cell. In some cases, the CAR contains an extracellular domain (ectodomain), which contains an antigen-binding domain, a stalk region, a transmembrane domain, and an intracellular (endodomain) domain. In some cases, the intracellular domain further comprises one or more intracellular signaling domains. In some cases, the CAR described in this application contains an antigen-binding domain, a stem region, a transmembrane domain, one or more co-stimulatory domains, and a signaling domain for T cell activation.

[000208] In embodiments, the CAR of the present invention comprises a target-specific binding element, otherwise referred to as an antigen-binding fragment. In embodiments, the CAR of the present invention is modified to target a tumor antigen of interest by modifying the target antigen-binding fragment that specifically binds to a predetermined antigen in the tumor cell. As used herein, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with hyperproliferative disorder" refers to antigens that are common to specific hyperproliferative disorders such as cancer.

[000209] An antigen-binding domain may comprise complementarity-determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen-binding fragments thereof. The complementarity determining region (CDR) is a short amino acid sequence found in the variable domains of antigen receptor proteins (e.g., immunoglobulin and T-cell receptor) that adopts a structure that is complementary to that of the antigen and therefore confers receptor specificity for that specific antigen. Each antigen receptor polypeptide chain may contain three CDRs (CDR1, CDR2 and CDR3). In some cases, the antigen binding domain contains F(ab') ₂ , Fab', Fab, Fv or scFv. In some cases, the antigen binding domain is scFv. In some cases, the antigen binding domain is a Fab. In some cases, the antigen binding domain is Fab'. In some cases, the antigen-binding domain is F(ab') ₂ . In some cases, the antigen binding domain is Fv.

[000210] In some embodiments, the CAR described herein comprises an antigen-binding domain that binds to an epitope on CD19, CD20, CD33, CD44, BCMA, CD123, EGFRvIII, folate receptor α, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesotheline, CD22, EGFR, MUC-1, MUC-16, MAGE- A1, h5T4, PSMA, TAG-72 or VEGF-R2. According to some embodiments of the present invention, the CAR described in this application contains an antigen-binding domain that binds to an epitope on CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE-A1, h5T4, PSMA, TAG-72 , EGFRvIII, CD123 and VEGF-R2. In some embodiments, the CAR described herein contains an antigen-binding domain that binds to an epitope on CD19 or CD33. In some cases, the CAR described in this application contains an antigen-binding domain that binds to an epitope on CD19. In some cases, the CAR described in this application contains an antigen-binding domain that binds to an epitope on CD33. According to additional embodiments of the present invention, the CAR or chimeric receptor or antigen-binding polypeptide described herein comprises a self-antigen or antigen-binding region that binds to an epitope on HLA-A2, oligodendrocyte myelin glycoprotein (MOG), factor VIII (FVIII), MAdCAMI, SDF1 or type II collagen.

[000211] In some embodiments, the polynucleotides, polypeptides, and methods described herein can be used to treat a hyperproliferative disease, such as cancer, an autoimmune disease, or to treat an infection, such as a viral, bacterial, or parasitic infection. In some aspects, an antigen is an antigen that is elevated in cancer cells, in autoimmune cells, or in cells that are infected with a virus, bacteria, or parasite. Pathogens that may be targeted include, but are not limited to, pathogens such as Plasmodium, Trypanosome, Aspergillus, Candida, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, HSV, HPV, RSV, EBV, CMV, JC virus, VC virus or Ebola virus. Autoimmune diseases may include graft-versus-host disease, rheumatoid arthritis, lupus, celiac disease, Crohn's disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis, optic neuromyelitis, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, or autoimmune uveitis.

[000212] A pathogen recognized by a CAR may be essentially any kind of pathogen, but in some embodiments of the present invention, the pathogen is a fungus, bacteria, or virus. Examples of viral pathogens include members of the Adenoviridae families, Epstein-Barr virus (EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, VC virus, HPV, HSV, HHV virus family, hepatitis virus 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, Shigella, Campylobacter, Staphylococcus, Helicobacter, E, coil, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella. In some embodiments of the present invention, the pathogen recognition receptor Dectin-1 can be used to generate a CAR that recognizes a carbohydrate structure on the cell wall of fungi such as Aspergillus. According to another embodiment of the present invention, CARs can be generated from an antibody that recognizes viral determinants (eg, glycoproteins from CMV and Ebola) to stop viral infections and pathology.

[000213] According to some embodiments of the present invention, a "stem" region or a "spacer" or "hinge" region is used to connect an antigen-binding domain to a transmembrane domain. In some instances, a "stem domain" or "stem region" comprises any oligonucleotide or polypeptide whose function is to link a transmembrane domain to either an extracellular domain or a cytoplasmic domain in a polypeptide chain. In some embodiments of the present invention, it is flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. In some cases, the stem region contains a hinge region from IgG1. In other instances, the stem region contains a CH2CH3 immunoglobulin region and optionally portions of CD3. In some cases, the stem region contains a CD8α hinge, a 12 amino acid IgG4-Fc hinge (ESKYGPPCPPCP (SEQ ID NO: 225)) or lgG4 hinges described in WO/2016/073755.

[000214] The transmembrane domain can be from either a natural or a synthetic source. If the source is natural, the domain may be from any membrane-bound or transmembrane protein. Suitable transmembrane domains may include the transmembrane region(s) of the alpha, beta, or zeta chain of the T cell receptor; or a transmembrane region of CD28, CD3-epsilon, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In another embodiment, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In some embodiments of the present invention, the triplet of phenylalanine, tryptophan and valine is located at one or both ends of the synthetic transmembrane domain. Optionally, a short oligonucleotide or polypeptide linker, according to some embodiments of the present invention, from 2 to 10 amino acids in length, can form a link between the transmembrane domain and the CAR cytoplasmic signaling domain. In some embodiments of the present invention, the linker is a glycine-serine linker. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain or a CD3ζ transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In other embodiments of the present invention, the transmembrane domain comprises a CD3ζ transmembrane domain. In other embodiments of the present invention, the transmembrane domain comprises a dimerization transmembrane domain.

[000215] An intracellular domain may contain one or more costimulatory domains. Exemplary co-stimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof. In some cases, the CAR described in this application contains one or more or two or more costimulatory domains selected from CD8, CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or their fragment or combination. In some cases, the CAR described in this application contains one or more or two or more costimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, OX40 (CD134), or a fragment or combination thereof. In some cases, the CAR described in this application contains one or more or two or more costimulatory domains selected from CD8, CD28, 4-1 BB (CD137), or a fragment or combination thereof. In some cases, the CAR described in this application contains one or more or two or more co-stimulatory domains selected from CD28, 4-1 BB (CD137), or a fragment or combination thereof. In some cases, the CAR described in this application contains costimulatory domains of CD28 and 4-1 BB (CD137) or their respective fragments. In some cases, the CAR described in this application contains co-stimulatory domains of CD28 and OX40 (CD134) or their respective fragments. In some cases, the CAR described in this application contains CD8 and CD28 co-stimulatory domains or their respective fragments. In some cases, the CAR described in this application contains CD28 co-stimulatory domains or a fragment thereof. In some cases, the CAR described in this application contains co-stimulatory domains 4-1 BB (CD137) or a fragment thereof. In some cases, the CAR described in this application contains co-stimulatory domains of OX40 (CD134) or a fragment thereof. In some cases, the CAR described in this application contains CD8 co-stimulatory domains or a fragment thereof.

[000216] The intracellular signaling domain, also known as the cytoplasmic domain, of the CAR of the present invention causes activation of at least one of the normal effector functions of the immune cell into which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be a cytolytic activity or ancillary activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that signals an effector function and directs the cell to perform a specialized function. Although it is usually possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact strand, provided that it signals the effector function. Thus, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to signal an effector function. According to some embodiments of the present invention, the intracellular domain further comprises a signaling domain for activating T cells. In some cases, the signaling domain for T cell activation contains a domain derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b or CD66d. In some cases, the signaling domain for T cell activation contains a domain derived from CD3ζ.

[000217] The term "functional part", when applied to a CAR, refers to any part or fragment of a CAR according to the present invention, and this part or fragment retains the biological activity of the CAR of which it (it) is (the original CAR). With respect to the nucleic acid sequence encoding the original CAR, the nucleic acid sequence encoding the functional portion of the CAR may encode a protein containing, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95 % or more of the original CAR.

[000218] As used herein, the term "functional variant" refers to a polypeptide or protein that has substantial or significant sequence identity or similarity to a reference polypeptide and retains the biological activity of the reference polypeptide of which it is a variant. Functional variants include, for example, those CAR variants described herein (original CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a greater extent as the original CAR. With respect to a nucleic acid sequence encoding a parent CAR, a nucleic acid sequence encoding a functional CAR variant may be, for example, about 10%, about 25%, about 30%, about 50%, about 65%, about 80%, about 90%, about 95%, or about 99% identical to the nucleic acid sequence encoding the original CAR.

[000219] The polynucleotide constructs disclosed herein can be co-expressed with CAR in a modified cell. In some embodiments of the present invention, the polypeptide construct and the CARs may be encoded by the same transcript. An advantage of encoding a polypeptide construct containing the truncated variant and CAR in the same transcript is that the modified cell producing the CAR protein is also reasonably likely to produce the polypeptide construct. Accordingly, if an intervention is required during immunotherapy to reduce CAR expression (e.g., to reduce the side effects of therapy), a modified cell expressing polypeptide constructs in conjunction with CAR can be activated to respond in a relatively short period of time to the introduction of exogenous antibodies that target cell labels provided with truncated variants disclosed in this application.

CD19-specific CARs

[000220] CD19 is a cell surface glycoprotein from the immunoglobulin superfamily. In some cases, CD19 has been found in solid tumors such as pancreatic cancer, liver cancer, and prostate cancer.

[000221] In some embodiments, the CAR antigen-binding fragment described herein is specific for CD19. CD19-specific CAR expressed on the cell surface can redirect T cell specificity to human CD19. In embodiments, the antigen-binding domain comprises a single chain antibody fragment (scFv) comprising the light chain variable region (VL) and the heavy chain variable region (VH) of an anti-CD19 monoclonal antibody specific for the target antigen, joined by a flexible linker, such as a glycine-serine linker. or Whitlow linker. In embodiments, the scFvs are SJ25C1 and/or FMC63. In embodiments, the scFv is humanized. According to some embodiments of the present invention, the antigen-binding fragment may contain VH and VL, which are directed connected, for example, from the N - to the C-terminus, VH-linker-VL or VL-linker-VH.

[000222] Some embodiments of the present invention described in this application include CD19-specific CAR, in which the antigen-binding domain contains scFv that binds CD19. In some cases, the antigen binding domain recognizes an epitope on CD19.

[000223] 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). Some embodiments of the invention described herein include 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). In some cases, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000224] Some embodiments of the invention described herein include a CD19-specific CAR-T cell comprising an scFv antigen-binding domain, said antigen-binding domain recognizing an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno THErapeutics). In some cases, the C019-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000225] In some embodiments, the CD19-specific CAR-T cell described herein comprises an anti-CD19 antibody as described in US20160152723.

[000226] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.). Some embodiments of the invention described herein include a CD19-specific CAR-T cell in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000227] Some embodiments of the invention described herein include a CD19-specific CAR-T cell comprising an scFv antigen-binding domain, said antigen-binding domain recognizing an epitope on CD19 that is also recognized by KTE-C19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000228] In some embodiments, the CD19-specific CAR-T cell described herein comprises an anti-CD19 antibody as described in WO 2015187528, or a fragment or derivative thereof.

[000229] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). Some embodiments of the invention described herein include a CD19-specific CAR-T cell in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, a CD19-specific CAR-T cell further comprises a transmembrane domain selected from CD8-alpha transmembrane domain or CD3ζ transmembrane domain one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10 , DAP12, OX40 (CD134) or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000230] Some embodiments of the invention described herein include a CD19-specific CAR-T cell comprising an scFv antigen-binding domain, said antigen-binding domain recognizing an epitope on CD19 that is also recognized by CTL019. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ. In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellectis). Some embodiments of the invention described herein include a CD19-specific CAR-T cell in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some cases, a CD19-specific CAR-T cell further comprises a transmembrane domain selected from CD8-alpha transmembrane domain or CD3ζ transmembrane domain one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10 , DAP12,OX40 (CD134) or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000231] Some embodiments of the invention described herein include a CD19-specific CAR-T cell comprising an scFv antigen-binding domain, said antigen-binding domain recognizing an epitope on CD19 that is also recognized by UCART19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000232] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by BRX-401 (Bellicum). Some embodiments of the invention described herein include a CD19-specific CAR-T cell in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BRX-401. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000233] Some embodiments of the invention described herein include a CD19-specific CAR-T cell comprising an scFv antigen-binding domain, said antigen-binding domain recognizing an epitope on CD19 that is also recognized by BRX-401. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000234] In some instances, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi-aventis), MOR208 (Morphosys AG/Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumabpapatoxom (National Cancer Institute), XmAb 5871 (Amgen/Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed). In some cases, the CD19-specific CAR further comprises a transmembrane domain selected from CD8-alpha transmembrane domain or CD3ζ transmembrane domain one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000235] Some embodiments described herein include a CD19-specific CAR-T cell in which the antigen-binding domain contains F(ab') ₂ , Fab', Fab, Fv, or scFv. In some cases, the antigen binding domain recognizes an epitope on CD19. In some cases, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi-aventis), MOR208 (Morphosys AG/Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics). ), B4 (or DI-B4) (Merck Serono), taplitumabpapatoxom (National Cancer Institute), XmAb 5871 (Amgen/Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed). In some instances, the CD19-specific CAR T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000236] In some instances, the CD19-specific CAR-T cell described herein contains an scFv antigen-binding domain, said antigen-binding domain recognizing an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi- aventis), MOR208 (Morphosys AG/Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumabpapatoxom (National Cancer Institute), XmAb 5871 (Amgen/ Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed). In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1 BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[000237] According to one embodiment of the present invention, a CAR-encoding polynucleotide contains a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence, selected from the list consisting of SEQ ID NO: 169 (encoding CD19-CD3ζ CAR), SEQ ID NO: 171 (encoding CD19-CD137-CD3ζ CAR), SEQ ID NO: 173 (encoding CD19-CD28-CD3ζ CAR), and SEQ ID NO: 175 (encoding CD19-CD28-CDζ CAR, additionally containing an IgG4 Fc spacer). According to one implementation variant of the present invention, the amino acid sequence containing CAR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence selected from the list consisting of SEQ ID NO: 170 (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, additionally containing an Fc IgG4 spacer).

[000238] In some embodiments, a polynucleotide disclosed herein may comprise a codon-optimized cDNA sequence encoding an anti-CD19 CAR and a cleavable T2A linker that binds to a cell surface polypeptide (e.g., HER1t). For example, cytotoxic T lymphocytes can be modified to express a CD19-specific chimeric antigen receptor (CAR) that signals from a cytoplasmic co-stimulatory domain (CD28) fused to the CD3ζ; cytoplasmic domain. The polypeptide may further comprise a C-terminal cleavable 2A linker followed by, for example, a cell extracellular tag containing, in some embodiments of the present invention, a truncated human HER1 (HER1t), a truncated CD20 (CD20t), a truncated CD52 (CD52t), or truncated LNGFR (LNGFRt). According to other embodiments of the present invention, the polypeptide may comprise a polypeptide construct containing a truncated variant preceding the CD19-specific CAR (eg, via a cleavable P2A linker).

[000239] The present invention provides polynucleotides and polypeptides encoding CAR and any polypeptide construct described herein. In one embodiment of the present invention, a polypeptide construct comprising an anti-CD19 CAR, a cleavable T2A linker, and a cell surface polypeptide is encoded by a polynucleotide comprising a nucleotide sequence that 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: 181 (CD19-CD28-CD3ζ CAR. P2A. Ig. HER1t1 kappa signal peptide of SEQ ID NO: 56) and SEQ ID NO: 185 (CD19-CD137-CD3ζ CAR.E2A. Ig.HER1t7 kappa signal peptide from SEQ ID NO: 68).

[000240] According to one embodiment of the present invention, a polypeptide construct comprising a CAR directed against CD19, a cleavable T2A linker, and a cell surface polypeptide of 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: 179 (CD19-CD137-CD3ζ CAR.T2A.Ig.HER1t kappa signal peptide of SEQ ID NO: 55), SEQ ID NO: 180 (CD19 -CD137-CD3ζ CAR.T2A.Ig.HER1t kappa signal peptide from SEQ ID NO: 55), SEQ ID NO:182 (CD19-CD28-CD3ζ CAR.P2A.Ig.HER1t1 kappa signal peptide from SEQ ID NO:57) , SEQ ID NO: 183 (Ig.HER1t1.P2A kappa signal peptide. CD8α.CD19-CD28-CD3ζ CAR signal peptide, where HER1t1 is from SEQ ID NO: 57), SEQ ID NO: 184 (CD19-CD28-CD3ζ CAR. furin-T2A. Ig.HER1t1 kappa signal peptide of SEQ ID NO: 57), SEQ ID NO: 186 (CD19-CD137-CD3ζ CAR.E2A. Ig.HER1t7 kappa signal peptide of SEQ ID NO: 69), SEQ ID NO : 187 (CD19-CD28-CD3ζ CAR. furin-T2A. Ig. HER1t8 kappa signal peptide from SEQ ID NO: 73), SEQ ID NO: 188 (CD19-CD137-CD3ζ CAR. E2A. Ig. HER1t8 kappa signal peptide from SEQ ID NO: 73), SEQ ID NO: 189 (CD19-CD28-CD3ζ CAR. furin-T2A. Ig. HER1t9 kappa signal peptide of SEQ ID NO: 77), SEQ ID NO: 190 (CD19-CD137-CD3ζ CAR .E2A. Ig. HER1t9 kappa signal peptide of SEQ ID NO: 77), SEQ ID NO: 191 (CD19-CD28-CD3ζ CAR. furin-T2A. Ig. HER1t10 kappa signal peptide of SEQ ID NO: 81), SEQ ID NO: 192 (CD19-CD137-CD3ζ CAR.E2A.Ig.HER1t10 kappa signal peptide of SEQ ID NO: 81), SEQ ID NO: 194 (CD19-CD28-CD3ζ CAR.P2A.CD20), SEQ ID NO: 195 (CD19-CD28-CD3ζ CAR.P2A.CD20t1 of SEQ ID NO: 109), SEQ ID NO: 196 (CD19-CD28-CD3ζ CAR.P2A.CD20t4 of SEQ ID NO: 115), SEQ ID NO: 197 (CD52t3 .P2A. CD8α.CD19-CD28-CD3ζ CAR signal peptide, where CD52t3 from SEQ ID NO: 149) and SEQ ID NO: 198 (Ig.LNGFRt4 kappa signal peptide. P2A. CD8a.CD19-CD28-CD3ζ CAR signal peptide, where LNGFRt4 from SEQ ID NO: 165).

Modified T cell receptor (TCR)

[000241] According to some variants of implementation of the present invention, the chimeric receptor encoded by the polynucleotide described in this application contains a modified T-cell receptor. The T cell receptor (TCR) consists of two chains (αβ or γδ) that pair on the surface of the T cell to form a heterodimeric receptor. In some cases, the αβ TCR is expressed on most T cells in the body and is known to be involved in the recognition of specific MHC-restricted antigens. Each α- and β-chain consists of two domains: a constant domain (C), which attaches the protein to the cell membrane and is associated with invariant subunits of the CD3 signaling apparatus; and a variable domain (V) that provides antigen recognition with six loops called complementarity determining regions (CDRs). In some cases, each of the V-domains contains three CDRs; for example, CDR1, CDR2 and CDR3, while CDR3 is a hypervariable region. These CDRs interact with a complex formed by an antigenic peptide associated with a protein encoded by the major histocompatibility complex (pepHCHC) (e.g., HLA-A complex, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA -DQB1, HLA-DRA or HLA-DRB1). In some cases, the constant domain further comprises a connecting region that connects the constant domain to the variable domain. In some cases, the beta chain additionally contains a short region of diversity, which is part of the connecting region.

[000242] In some cases, such TCRs are able to interact with a specific tumor antigen, for example, NY-ESO, Mage A3, Titin. In other instances, such TCRs are capable of interacting with specific neoantigens expressed in the patient's tumor (ie, patient-specific somatic, non-synonymous mutations expressed by tumors). In some cases, the affinity of modified TCRs can be increased.

[000243] According to some embodiments of the present invention, the TCR is described using the International Database for Immunogenetics (IMGT) TCR nomenclature, as well as references to TCR sequences from the public IMGT database. For example, there may be several types of alpha chain variable regions (Vα) and several types of beta chain variable regions (Vβ) distinguished by their framework, CDR1, CDR2 and CDR3 sequences. As such, a Vα type may be referred to in the IMGT nomenclature by a unique TRAV number. For example, "TRAV21" defines a TCR Vα region having a unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by an amino acid sequence that is conserved across TCRs, but which also contains an amino acid sequence that varies across TCRs. Similarly, "TRBV5-1" defines a TCR Vβ region having a unique framework and CDR1 and CDR2 sequences, but only with a partially defined CDR3 sequence.

[000244] In some instances, the beta chain diversity region is referred to in the IMGT nomenclature as the abbreviation TRBD.

[000245] In some cases, unique sequences defined using the IMGT nomenclature are widely known and available to those working in the TCR field. For example, they can be found in the IMGT public database and in "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[000246] According to some embodiments of the present invention, the αβ-heterodimeric TCR, for example, is transfected as full length chains having both cytoplasmic and transmembrane domains. In some cases, the TCRs contain an introduced disulfide bond between the residues of the respective constant domains, as described, for example, in WO 2006/000830.

[000247] In some cases, the TCRs described in this application are in single strand format, for example, see WO 2004/033685. Single chain formats include αβ TCR types Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-Cβ, where Vα and Vβ represent are TCR α and β variable regions, respectively, Cα and Cβ are TCR α and β constant regions, respectively, and L is a linker sequence. According to certain embodiments of the present invention, the single chain TCRs of the present invention may have an introduced disulfide bond between the residues of the respective constant domains, as described in WO 2004/033685.

[000248] The TCR described herein may be associated with a detectable label, therapeutic agent, or PK-modifying fragment.

[000249] Exemplary detectable labels for diagnostic purposes include, but are not limited to, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

[000250] The present invention provides polynucleotides and polypeptides encoding a TCR and any polypeptide construct described herein. In some embodiments, a polynucleotide disclosed herein may comprise a codon-optimized cDNA sequence encoding a TCR and a cleavable T2A linker that binds to a cell surface polypeptide (eg, HER1t). For example, a cytotoxic T lymphocyte can be modified to express a TCR polypeptide that further comprises a C-terminal cleavable 2A linker followed by, for example, a cell extracellular label comprising, in some embodiments of the present invention, truncated human HER1 (HER1t), truncated CD20 (CD20t), truncated CD52 (CD52t), or truncated LNGFR (LNGFRt). According to other embodiments of the present invention, the polypeptide may comprise a polypeptide construct containing a truncated variant preceding the TCR (eg, attached via a cleavable P2A linker).

Cytokines

[000251] According to the present invention, polynucleotides encoding polypeptide cell tags and a cytokine, or a variant or derivative thereof, as well as methods and systems containing the above, are provided. A cytokine is a type of small proteins with a mass of approximately 5-20 kDa that are involved in cell signaling. According to some embodiments of the present invention, cytokines may be membrane bound or may be secreted. In other embodiments of the present invention, the cytokines may be intracellular. In some cases, cytokines include chemokines, interferons, interleukins, colony stimulating factors or tumor necrosis factors. In some embodiments of the present invention, chemokines act as a chemoattractant to direct cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. CCL5 (RANTES) and CCL28; CXC subfamilies: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17; XC subfamilies: XCL1 and XCL2; and subfamilies CX3C: CX3CL1.

[000252] Interferons (IFNs) contain type I interferon (eg, IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), type II interferon (eg, IFN-γ), and type III interferon. According to some embodiments of the present invention, IFN-α is further classified into about 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[000253] Interleukins are expressed by leukocytes or white blood cells and stimulate the development and differentiation of T and B lymphocytes and hematopoietic cells. Exemplary interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36.

[000254] In some embodiments, the interleukin comprises mbIL-15. In some embodiments, mbIL-15 is a membrane-bound chimeric IL-15 that can be co-expressed with the modified effector cell described herein. In some embodiments, mbIL-15 comprises full-length IL-15 (eg, native IL-15 polypeptide) or a fragment or variant thereof hybridized in frame with full-length IL-ISRα, a functional fragment or variant thereof. In some cases, IL-15 is indirectly linked to IL-ISRα via a linker. In some cases, mbIL-15 is the one described in Hurton et al., "TetHERed IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells," PNAS 2016. According to one embodiment of the present of the invention, mbIL-15 is encoded by a polynucleotide containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 177. According to one in an embodiment of the present invention, mbIL-15 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 178.

[000255] According to another aspect, the interleukin may contain IL-12. In some embodiments, IL-12 is single chain IL-12 (ocIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, intercalated IL-12. In some instances, IL-12 variants are those described in WO 2015/095249, WO 2016/048903, WO 2017/062953, all of which are incorporated by reference in their entirety.

[000256] Tumor necrosis factors (TNF) are a group of cytokines that modulate apoptosis. In some cases, the TNF family contains approximately 19 members, including, but not limited to, TNFα, lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), gp39 T-cell antigen (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L and TNF-linked apoptosis inducing ligand (TRAIL).

[000257] Colony stimulating factors (CSFs) are secreted glycoproteins that interact with receptor proteins on the surface of hematopoietic stem cells, which subsequently modulate cell proliferation and differentiation into specific blood cell types. In some cases, CSF includes macrophage colony stimulating factor, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or promegapoietin.

[000258] According to some embodiments of the present invention, a cytokine is a membrane-bound cytokine that is co-expressed with a chimeric antigen receptor and/or TCR described herein.

[000259] According to some variants of implementation of the present invention, one or more of the methods described in this application further include the introduction of a cytokine. In some cases, the cytokine includes a chemokine, interferon, interleukin, colony stimulating factor or tumor necrosis factor. In some instances, one or more of the methods described herein further comprise administering a cytokine, a selected chemokine, an interferon, an interleukin, a colony stimulating factor, or a tumor necrosis factor. In some cases, one or more of the methods described in this application further include the introduction of a cytokine selected from IL-2, IL-7, IL-12, IL-15, IL-21, IFN-γ or TNF-α.

[000260] In some embodiments, a polynucleotide disclosed herein may comprise a codon-optimized cDNA sequence encoding a cytokine and a cleavable T2A linker that binds to a cell surface polypeptide (eg, HER1t). For example, a cytotoxic T lymphocyte can be modified to express a polypeptide containing a cytokine and further comprising a cleavable 2A linker followed by, for example, an extracellular label of the cell containing, in some embodiments of the present invention, truncated human HER1 (HER1t), truncated CD20 (CD20t), truncated CD52 (CD52t), or truncated LNGFR (LNGFRt). According to other embodiments of the present invention, the polypeptide may comprise a polypeptide construct containing a truncated variant preceding the cytokine (eg, attached via a cleavable P2A linker).

[000261] The present invention provides polynucleotides and polypeptides encoding a cytokine and any polypeptide construct described herein. In one embodiment of the present invention, a polypeptide construct comprising a cytokine, a cleavable T2A linker, and a cell surface polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identical. SEQ ID NO: 193 (membrane-bound IL-15.T2A.HER1t1 from SEQ ID NO: 57).

IRES and linkers

[000262] Also disclosed are constructs containing linkers and IRES elements to facilitate the expression and functionality of the polynucleotides and polypeptides described in this application.

IRES elements

[000263] As used herein, the term "internal ribosome entry site (IRES)" may be intended to refer to an internal ribosome entry site. In a vector containing the IRES sequence, the first gene can be translated by a cap-dependent ribosome scanning mechanism with its own 5'-UTR, while translation of the subsequent gene can be done by directly recruiting the ribosome to the IRES in a cap-independent manner. The IRES sequence can provide eukaryotic ribosome binding and translation initiation without binding to the 5'-capped end. An IRES sequence can allow the expression of multiple genes from a single transcript (Mountford and Smith 1995).

[000264] As used herein, the term "CAP" or "cap" refers to a modified nucleotide, typically 7-methylguanosine, fused 3' to 5' (7meG-ppp-G) to the 5' end of a eukaryotic mRNA, which is an essential element in normal translation initiation pathway during protein expression from this mRNA.

[000265] In certain cases, the IRES region may be derived from a virus, such as picornavirus, encephalomyocarditis virus, hepatitis C virus IRES sequence. In other cases, the IRES sequence may be derived from encephalomyocarditis virus. As used herein, the term "EMCV" or "encephalomyocarditis virus" refers to an isolate of any member or strain of an encephalomyocarditis virus species from a genus belonging to the Picornaviridae family. Examples include: EMCV-R virus strain (Rueckert), Columbia-SK virus. In some instances, a cellular IRES element, such as the IRES of eukaryotic initiation factor 4G, immunoglobulin heavy chain binding protein, c-myc proto-oncogene, vascular endothelial growth factor, fibroblast growth factor-1, or any combination or modification thereof, may be used. In some cases, the cellular IRES may have increased gene expression compared to the viral IRES.

[000266] The vector may use a viral or cellular IRES sequence, or a combination thereof. IRES can be derived from encephalomyocarditis virus (EMCV) or poliovirus (PV). In some instances, the IRES element is selected from the group consisting of poliovirus (PV), encephalomyelitis virus (EMCV), foot-and-mouth disease virus (FMDV), porcine teshovirus-1 (PTV-1), achivirus (AiV), Seneca Valley virus (SW), hepatitis C virus (HCV), classical swine fever virus (CSFV), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-1 (HIV-1), Moloney murine leukemia virus (MoMLV), feline immunodeficiency virus (FIV ), murine mammary tumor virus (MMTV), latent human cytomegalovirus (pUL138), Epstein-Barr virus (EBNA-1), Marek's disease herpesvirus (MDV RLORF9), polycistronic 19S SV40 (SV40 19S), Rhopalosiphum padi virus (RhPV) , cricket paralysis virus (CrPV), Ectropis obliqua picornium-like virus (EoPV), Plautia stali intestinal tract virus (PSIV), triatomy virus (TrV), bee paralysis dicystrovirus (IAPV, KBV), black currant reversion virus (BRV), interruption virus pelargonium flower virus (PFBV), hibiscus ringspot virus (HCRSV), cruciferous tobamovirus (CrTMV), potato leafroll polerovirus (PLRV), tobacco etch virus (TEV), Giardia lamblia virus (GLV), Leishmania RNA virus 1 (LRV) -1) and their combinations or modifications. In some cases, the IRES is selected from the group consisting of Apaf-1, XIAP, HIAP2/C-IAP1, DAP5, Bcl-2, c-myc, CAT-1, INR, differentiation LEF-1, PDGF2, HIF-1a, VEGF , FGF2, BiP, BAG-1, CIRP, p53, SHMT1, PITSLREp58, CDK1, Rpr, hid, hsp70, grim, ski, Antennapedia, dFoxO, dlnR, Adh-Adhr, HSP101, ADH, URE-2, GPR1, NCE102 , YMR181a, MSN1, BOI1, FLO8, GIC1 and any combination or modification thereof.

[000267] According to certain embodiments of the present invention, the IRES is an EMCV IRES containing a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical to the nucleotide sequence of SEQ ID NO: 49.

[000268] If an IRES element is included between two open reading frames (ORFs), translation initiation may occur via a canonical 5'-m7GpppN cap dependent mechanism in the first ORF and a cap independent mechanism in the second ORF after the IRES element.

[000269] In some instances, genes can be connected via an internal ribosome entry site (IRES). IRES can provide simultaneous expression of multiple genes. For example, an IRES sequence can provide multiple proteins from a single mRNA transcript. Ribosomes can bind to IRES in a 5'-cap independent manner and initiate translation.

[000270] In some cases, the IRES sequence may be 500 bp or so. The IRES sequence can be from 300 base pairs to 1000 base pairs. For example, an IRES may be 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 bp in length.

[000271] In some cases, the expression of the 3'-terminal gene in a vector containing the IRES sequence can be reduced. For example, a gene located after the IRES sequence may have reduced expression compared to the gene preceding the IRES sequence. The reduction in expression can range from 1% to 99% compared to the previous gene.

Linkers

[000272] According to some embodiments of the present invention, a polynucleotide linker can be used in a polynucleotide described in this application. The polynucleotide linker can be a double-stranded DNA segment containing target restriction sites that can be added to create end structures that are compatible with the vector containing the polynucleotide described in this application. In some cases, a polynucleotide linker can be used to modify vectors containing the polynucleotides described in this application. For example, modification of a vector containing a polynucleotide linker may be a change in the multiple cloning site or the addition of a polyhistidine tail. Polynucleotide linkers can also be used to adapt the ends of the blunt-ended DNA insert for cloning into a sticky-ended restriction enzyme-cleaved vector. The use of polynucleotide linkers may be more efficient than blunt-end ligation into the vector and may provide a way to release the insert from the vector in subsequent applications. In some cases, the insert may be a polynucleotide sequence encoding polypeptides suitable for therapeutic applications.

[000273] The polynucleotide linker may be an oligomer. The polynucleotide linker may be double stranded DNA, single stranded DNA, or a combination thereof. In some cases, the linker may be RNA. In some cases, a polynucleotide linker can be ligated into a vector containing a polynucleotide described in this application using T4 ligase. In order to facilitate ligation, an excess of polynucleotide linkers may be added to the composition containing the insert and the vector. In some cases, the insert and the vector are pre-treated before the introduction of the linker. For example, pre-treatment with methylase can prevent unwanted cleavage of the DNA insert.

[000274] In embodiments, the polynucleotide linker comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical to the nucleotide sequence of region 2A (P2A) porcine teshovirus-1 (SEQ ID NO: 41); region 2A (E2A) of equine rhinitis virus A (SEQ ID NO: 43); regions 2A (T2A) of Thosea asigna virus (SEQ ID NO: 45); or FMDV region 2A (F2A) (SEQ ID NO: 47).

[000275] According to certain embodiments of the present invention, two or more polypeptides encoded by a polynucleotide described in this application can be separated by an intermediate sequence encoding an intermediate linker polypeptide. In this application, the term "intermediate linker polypeptide", referring to an amino acid sequence separating two or more polypeptides encoded by a polynucleotide, is different from the term "peptide linker", which refers to an amino acid sequence that is optionally included in the polypeptide construct disclosed in this application, to connect the transmembrane domain to a cell surface polypeptide (eg, containing a truncated version of the native polypeptide). In certain instances, the intermediate linker polypeptide is a cleavage-sensitive intermediate linker polypeptide. In some embodiments of the present invention, the polypeptides of interest are expressed as fusion proteins linked by a cleavage-sensitive intermediate linker polypeptide. According to certain embodiments of the present invention, the cleavage-sensitive intermediate(s) linker(s) polypeptide(s) may be any one or more of: F/T2A, T2A, p2A, 2A, GSG-p2A, GSG linker (SEQ ID NO: 16) and furinlink variants.

[000276] In embodiments, the intermediate linker polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical to the amino acid sequence of the region 2A (P2A) porcine teshovirus-1 (SEQ ID NO: 41); region 2A (E2A) of equine rhinitis virus A (SEQ ID NO: 44); regions 2A (T2A) of Thosea asigna virus (SEQ ID NO: 46); or FMDV region 2A (F2A) (SEQ ID NO: 48).

[000277] In some cases, the 2A viral sequence can be used. Elements 2A can be shorter than IRES and contain from 5 to 100 base pairs. In some cases, the 2A sequence may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides in length. 2A-linked genes can be expressed in a single open reading frame, and "self-cleavage" can occur co-translationally between the last two amino acids, GP, at the C-terminus of the 2A polypeptide, resulting in equal amounts of expressed proteins.

[000278] The 2A viral sequence may be about 20 amino acids. In some cases, the 2A viral sequence may contain the Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro consensus motif (SEQ ID NO: 229). The consensus motif sequence can act co-translationally.

For example, the formation of a normal peptide bond between glycine and proline residues can be prevented, which can lead to ribosome skipping and cleavage of the nascent polypeptide. This effect can result in equimolar levels of the products of several genes.

[000279] Peptide 2A can translate multiple proteins in a single open reading frame to produce a polypeptide that can subsequently be cleaved into individual polypeptides by a ribosome slip mechanism (Funston, Kallioinen et al. 2008). In some embodiments of the present invention, sequence 2A may include: F/T2A, T2A, p2A, 2A, T2A, E2A, F2A and BmCPV2A, BmIFV2A, and any combination thereof.

[000280] In some cases, the vector may contain an IRES sequence and a 2A polynucleotide linker sequence. In other cases, the expression of several genes connected by 2A peptides can be facilitated by using a spacer sequence (GSG (SEQ ID NO: 16)) before the 2A peptides. In some cases, the designs may combine spacers, linkers, adapters, promoters, or combinations thereof. For example, the construct may contain a spacer (SGSG (SEQ ID NO: 18) or GSG (SEQ ID NO: 16)) and an intermediate polypeptide linker with a furin cleavage site (R-A-K-R (SEQ ID NO: 230)) with various 2A peptides. The spacer may be I-Ceui. In some cases, the linker may be modified. For example, the linker can be designed to impart chemical characteristics such as hydrophobicity. In some cases, at least two linker sequences may result in the same protein. In other cases, multiple linkers may be used in the vector.

[000281] In certain cases, the intermediate linker polypeptide may contain the amino acid sequence "RAKR (SEQ ID NO: 230)". In certain instances, the furin intermediate linker polypeptide may be encoded by a polynucleotide sequence containing "AGAGCTAAGAGG (SEQ ID NO: 231)".

[000282] In certain cases, the intermediate linker polypeptide may be a linker containing the sequence disclosed in the table below:

[000283] According to some variants of implementation of the present invention, the linker can be used in the polynucleotide described in this application. The linker may be a flexible linker, a rigid linker, an in vivo cleavable linker, or any combination thereof. In some instances, the linker can join functional domains together (as in flexible and rigid linkers) or release a free functional domain under in vivo conditions, as in in vivo cleavable linkers.

[000284] According to some embodiments of the present invention, polynucleotide linkers and intermediate linker polypeptides can improve biological activity, increase expression yield, and achieve targeted pharmacokinetic profiles.

[000285] In some cases, the sequence of the intermediate linker polypeptide described in this application may include a flexible linker. Flexible linkers can be used if the attached domain requires a certain degree of movement or interaction. Flexible linkers may be composed of small non-polar (eg, Gly) or polar (eg, Ser or Thr) amino acids. A flexible linker may have sequences primarily composed of regions of Gly and Ser residues ("GS" linker). An example of a flexible linker may have the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 221; eg, SEQ ID NO: 20 or 22). The length of this exemplary GS linker can be optimized by adjusting the copy number "n" to achieve an appropriate separation of the functional domains or to support the necessary interdomain interactions. In addition to GS linkers, other flexible linkers can be used for recombinant fusion proteins. In some cases, flexible linkers may also be rich in small or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility.

[000286] The flexible linkers included in the linker sequences described herein can be rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. Flexible linkers may be an appropriate choice if certain movements or interactions are desired for the domains of the fusion protein. In addition, although flexible linkers may not contain rigid structures, they can serve as a passive linker to maintain distance between functional domains. The length of the flexible linker can be adjusted to ensure proper folding or to achieve optimal biological activity of the fusion proteins.

[000287] In some cases, the intermediate linker polypeptide described in this application may additionally contain a rigid linker. A rigid linker can be used to maintain a fixed distance between polypeptide domains. Examples of rigid linkers are: alpha helix linkers, Pro rich sequence, (XP)n, X-Pro backbone (SEQ ID NO: 239), A(EAAAK)nA (SEQ ID NO: 240) (n=2- 5) as a few examples. Rigid linkers can exhibit relatively rigid structures by taking the form of α-helical structures or by containing a few Pro residues in some cases.

[000288] The intermediate linker polypeptide described in this application may be cleavable in some cases. In other cases, the intermediate linker polypeptide is not cleavable. Linkers that are not cleavable can covalently link functional domains together to function as one molecule during an in vivo process or an ex vivo process. The intermediate linker polypeptide may also be cleavable under in vivo conditions. A cleavable intermediate linker polypeptide can be introduced, for example, to release free functional domains under in vivo conditions. The intermediate linker polypeptide can be cleaved, for example, by reducing agents and proteases present. For example, disulfide bond reduction can be used to produce a cleavable intermediate linker polypeptide. In the case of a disulfide intermediate linker polypeptide, cleavage may result from the exchange of the disulfide with a thiol such as glutathione. In other cases, in vivo cleavage of the intermediate linker polypeptide in the recombinant fusion protein can also be carried out by proteases that can be expressed in vivo under pathological conditions (eg, cancer or inflammation), in specific cells or tissues, or restrictedly within certain cellular compartments. . In some instances, a cleavable intermediate linker polypeptide may provide targeted cleavage. For example, the specificity of many proteases may allow slower cleavage of the intermediate linker polypeptide in limited compartments. The cleavable intermediate linker polypeptide may also contain hydrazone, peptides, a disulfide or a thioether. For example, hydrazone can confer serum stability. In other cases, the hydrazone may provide cleavage in the acidic compartment. The acid compartment may have a pH of up to about 7. The linker may also include a thioether. The thioether may be non-reducible and/or may be engineered for intracellular proteolytic degradation.

[000289] The linker may be a modified linker. Methods for constructing linkers may be computational. In some cases, computational methods may include graphical techniques. Computational methods can be used to search for suitable peptides in libraries of three-dimensional peptide structures obtained from databases. For example, the Brookhaven Protein Data Bank (PDB) can be used to determine the spatial distance between selected linker amino acids.

[000290] In some embodiments, the present invention provides polynucleotides encoding a polypeptide construct comprising a furin polypeptide and a 2A polypeptide, wherein the furin polypeptide and the 2A polypeptide are connected by an intermediate linker polypeptide containing at least three hydrophobic amino acids. In some cases, at least three hydrophobic amino acids are selected from the list consisting of glycine (Gly) (G), alanine (Ala) (A), valine (Val) (V), leucine (Leu) (L), isoleucine (Ile ) (I), proline (Pro) (P), phenylalanine (Phe) (F), methionine (Met) (M), tryptophan (Trp) (W).

Expression of polypeptide constructs

[000291] According to some embodiments of the present invention, the polynucleotides and polypeptides described in this application can be constitutively expressed, for example, using constitutive promoters of viral and non-viral delivery systems (see below). According to other embodiments of the present invention, the polypeptide constructs, polynucleotides, and methods of the present invention may be implemented in a gene switch system. The term "gene switch" refers to the combination of a promoter-associated response element and, for example, an EcR-based system that, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are inserted. Tightly regulated inducible gene expression systems or gene switches can be used for various applications such as gene therapy, large-scale production of proteins in cells, high throughput cellular assays, functional genomics, and trait regulation in transgenic plants and animals. Such inducible gene expression systems may include ligand-inducible heterologous gene expression systems.

[000292] An early version of the EcR-based gene switch used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) polypeptides and was shown that in the presence of the steroid, ponasterone A, these receptors transactivate reporter genes in mammalian cell lines and in transgenic mice (Christopherson et al., 1992; No et al., 1996). Later, Suhr et al., 1998 showed that the non-steroidal ecdysone agonist tebufenoside induced high levels of reporter gene transactivation in mammalian cells with EcR Bombyx mori(BmEcR) in the absence of an exogenous heterodimeric partner.

[000293] International Patent Applications Nos. PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO 99/58155) disclose methods for modulating exogenous gene expression in which a DNA construct comprising an exogenous gene and a response element to ecdysone is activated with a second DNA construct containing the ecdysone receptor, which, in the presence of its ligand and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression. In this example, the ecdysone receptor was isolated from Drosophila melanogaster. Typically, such systems require the presence of a silent partner, preferably the retinoid X receptor (RXR), to ensure optimal activation. In mammalian cells, the insect ecdysone receptor (EcR) is able to heterodimerize with the mammalian retinoid X receptor (RXR), which allows it to be used to regulate the expression of target genes or heterologous genes in a ligand-dependent manner. International Patent Application No. PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silkworm Bombyx mori functions in mammalian systems without the need for an exogenous dimerization partner.

[000294] US Pat. No. 6,265,173 discloses that various members of the steroid/thyroid hormone receptor superfamily can be combined with the Drosophila melanogaster ultraspiracle (USP) receptor or fragments thereof containing at least a USP dimerization domain for use in a gene expression system. US Pat. No. 5,880,333 describes a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimeric system for use in plants in which a transactivation domain and a DNA binding domain are located on two different fusion proteins. In each of these cases, the transactivation domain and the DNA binding domain (either as a native EcR as in International Patent Application No. PCT/US98/14215 or as a modified EcR as in International Patent Application No. PCT/US97/05330) were are built into one molecule, and other heterodimeric partners, either USP or RXR, were used in their native state.

[000295] International Patent Application No. PCT/US01/0905 discloses an ecdysone receptor-based inducible gene expression system in which the DNA transactivation and binding domains are separated from each other by placing them on two different proteins, resulting in significantly reduced non-specific activity in the absence of a ligand and significantly increased activity, compared with non-specific activity, in the presence of a ligand. This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in PCT/US97/05330 and PCT/US98/14215. It is hypothesized that the two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcriptional activation domain into a more favorable position relative to the DNA-binding domain so that when the DNA-binding domain binds to the DNA-binding site on the gene, the transactivation domain activates the promoter more efficiently (see for example, US patent No. 5283173). The two-hybrid gene expression system contains two gene expression cassettes; the former encodes a DNA binding domain hybridized to a nuclear receptor polypeptide, and the latter encodes a transactivation domain hybridized to another nuclear receptor polypeptide. It is assumed that in the presence of a ligand there is an induction of a conformational change that stimulates the interaction of the first polypeptide with the second polypeptide, which leads to dimerization of the DNA-binding domain and the transactivation domain. Because the DNA binding domain and the transactivation domain are on two different molecules, non-specific activity in the absence of a ligand is greatly reduced.

[000296] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily and is classified as group H subfamily 1 (referred to herein as "group H nuclear receptors"). Members of each group share 40-60% amino acid identity in the E domain (ligand binding) (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97: 161-163). In addition to the ecdysone receptor, other group H members of the nuclear receptor subfamily 1 include: ubiquitous receptor (UR), orphan receptor 1 (OR-1), nuclear steroid hormone receptor 1 (NER-1), RXR-interacting protein 15 (RIP-15 ), hepatic X receptor β (LXRβ), steroid hormone receptor-like protein (RLD-1), hepatic X receptor (LXR), hepatic X receptor α (LXRα), farnesoid X receptor (FXR), interacting with protein 14 receptor (RIP-14) and farnesol receptor (HRR-1).

[000297] In some instances, the inducible promoter may be a small molecule ligand inducible two polypeptide ecdysone receptor gene switch, such as Intrexon's RHEOSWITCH® gene switch. In some cases, the gene switch may be selected from ecdysone receptor-based components as described, but not limited to, in any of the systems described in: PCT/US2001/009050 (WO 2001/070816); U.S. Patent Nos. 7,091,038; 7776587; 7807417; 8202718; PCT/US2001/030608 (WO 2002/029075); U.S. Patent Nos. 8105825; 8168426; PCT/1J52002/005235 (WO 2002/066613); US Application No. 10/468200 (US Publication No. 20120167239); PCT/US2002/005706 (WO 2002/066614); U.S. Patent Nos. 7531326; 8236556; 8598409; PCT/U52002/005090 (WO 2002/066612); US Pat. No. 8,715,959 (US Publication No. 20060100416); PCT/US2002/005234 (WO 2003/027266); U.S. Patent Nos. 7601508; 7829676; 7919269; 8030067; PCT/U52002/005708 (WO 2002/066615); US Application No. 10/468192 (US Publication No. 20110212528); PCT/US2002/005026 (WO 2003/027289); U.S. Patent Nos. 7,563,879; 8021878; 8497093; PCT/US2005/015089 (WO 2005/108617); US patent No. 7935510; 8076454; PCT/U52008/011270 (WO 2009/045370); US Application No. 12/241018 (US Publication No. 20090136465); PCT/US2008/011563 (WO 2009/048560); US Application No. 12/247738 (US Publication No. 20090123441); PCT/US2009/005510 (WO 2010/042189); US Application No. 13/123129 (US Publication No. 20110268766); PCT/US2011/029682 (WO 2011/119773); US Application No. 13/636473 (US Publication No. 20130195800); PCT/US2012/027515 (WO 2012/122025); and US Pat. No. 9,402,919, each of which is incorporated by reference in its entirety.

Modified effector cells

[000298] Effector cells modified to express one or more polypeptide constructs capable of functioning as cell labels in effector cells are provided.

[000299] As used herein, the term "T cell" or "T lymphocyte" is a type of lymphocyte that plays a major role in cellular immunity. They can be distinguished from other lymphocytes such as B cells and natural killer (NK) cells by the presence of a T cell receptor (TCR) on the cell surface.

[000300] According to some embodiments of the present invention, the modified effector cells are modified immune cells, which include T cells and/or natural killer cells. T cells or T lymphocytes are a subtype of white blood cells that are involved in cellular immunity. Exemplary T cells include helper T cells, cytotoxic T cells, TH17 cells, memory stem T cells (TSCM), naïve T cells, memory T cells, effector T cells, regulatory T cells, or natural killer T cells. T cells.

[000301] Helper T cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells and the activation of cytotoxic T cells and macrophages. In some cases, TH cells are known as CD4+ T cells due to the expression of the CD4 glycoprotein on the surface of the cells. Helper T cells are activated when they are presented with peptide antigens by class II MHC molecules, which are expressed on the surface of antigen presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in an active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate different types of immune responses. APC signaling directs T cells to specific subtypes. According to some variants of implementation of the present invention in the present application, the secreted cytokines may be recombinant.

[000302] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to an antigen associated with class I MHC molecules that are present on the surface of all nucleated cells. With the help of IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an energetic state, which prevents autoimmune diseases.

[000303] Memory T cells are a subgroup of antigen-specific T cells that persist long after infection has cleared. They multiply rapidly, producing large numbers of effector T cells upon repeated exposure to their cognate antigen, providing the immune system with a "memory" against past infections. Memory T cells include subtypes: stem memory T cells (TSCM), central memory T cells (TCM cells), and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells can express cell surface proteins CD45RO, CD45RA and/or CCR7.

[000304] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are involved in maintaining immunological tolerance. Their main role is to turn off T-cell mediated immunity by the end of the immune response and suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[000305] Natural killer T cells (NKT cells) mediate between the adaptive immune system and the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MCHC) molecules, NKT cells recognize a glycolipid antigen presented by a molecule called CD1d. Once activated, these cells can perform the functions attributed to both Th and Tc cells (ie, production of cytokines and release of cytolytic/cell-killing molecules). They are also able to recognize and destroy certain tumor cells and cells infected with herpes viruses.

[000306] Natural killer (NK) cells are a type of cytotoxic lymphocyte of the innate immune system. In some cases, NK cells provide first line protection against viral infections and/or tumor formation. NK cells can detect MHC presented on infected or cancerous cells, causing the release of cytokines, and subsequently induce lysis and apoptosis. NK cells can additionally detect stress cells in the absence of antibodies and/or MHC, resulting in a rapid immune response.

Virus-based delivery systems

[000307] The present invention provides delivery systems, such as virus-based systems, into which the nucleic acid described herein is inserted. Exemplary viral expression vectors include, but are not limited to, adeno-associated virus vectors, adenovirus vectors (e.g. adenovirus-based Reg.Cb system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus vectors ( for example, lentivirus pLPI from Life Technologies (Carlsbad, CA, USA)), retroviral vectors (eg pFB-ERV plus pCFB-EGSH), and herpes virus vectors. In one embodiment of the present invention, the viral vector is a lentiviral vector. Vectors derived from retroviruses, such as lentivirus, are suitable tools to achieve long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have an additional advantage such as low immunogenicity. In a further embodiment, the viral vector is an adeno-associated virus vector. In an additional embodiment, the viral vector is a retroviral vector. Typically, and in embodiments, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, restriction endonuclease convenience sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and patent US No. 6326193).

[000308] Additional suitable vectors include integrating expression vectors, which may randomly integrate into the DNA of the host cell or may contain a recombination site to allow for specific recombination between the expression vector and the chromosome of the host cell. Such integrating expression vectors can use endogenous expression control sequences from the chromosomes of the host cell to effect expression of the target protein. Examples of vectors that integrate in a site-specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, CA, USA) (e.g., pcDNATM5/FRT) or the cre-lox system, for example, which can be found in pExchange Master Vectors -6 from Stratagene (La Jolla, California, USA). Examples of vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when administered in the absence of T antigen) from Invitrogen (Carlsbad, CA, USA) and pCI or pFN10A (ACT) FLEXITM from Promega (Madison , Wisconsin, USA). Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. As a rule, they are located in the region of 30-110 bp. 5' from the start site, although it has recently been shown that a number of promoters also contain functional elements located 3' from the start site. The spacing between promoter elements is often not strict so that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be extended up to 50 bp before activity begins to decline. Apparently, depending on the promoter, individual elements can function either together or independently to activate transcription.

[000309] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it.

[000310] Another example of a suitable promoter is the human elongation factor 1-alpha 1 (hEF1a1) promoter. In embodiments, the vector construct comprising the CAR and/or TCR of the present invention contains functional variants of hEF1a1.

[000311] However, other constitutive promoter sequences can also be used, including, but not limited to, the simian early 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter ), the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also provided as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, the metallothioneine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[000312] In order to assess the expression of a CAR or TCR polypeptide or parts thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population cells to be transfected or infected with viral vectors. In other aspects, the selectable marker carrier may be a single DNA fragment and may be used in a co-transfection procedure. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to allow expression in host cells. Suitable selectable markers include, for example, antibiotic resistance genes such as neomycin resistance gene (neo) and ampicillin resistance gene and the like. In some embodiments of the present invention, a truncated epidermal growth factor receptor (HER1t) tag can be used as a selectable marker gene.

[000313] Reporter genes can be used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present or not expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is manifested through some readily detectable property, such as enzymatic activity. The expression of the reporter gene is assessed at the appropriate time after the introduction of the DNA into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or a green fluorescent protein gene (eg, Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be obtained using known techniques or obtained commercially. Typically, a construct with a minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and can be used to assess the ability of agents to modulate promoter-driven transcription.

[000314] In some embodiments, the vectors contain the hEF1a1 promoter to drive transgene expression, a bovine growth hormone polyA sequence to enhance transcription, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and LTR sequences derived from the pFUGW plasmid.

[000315] Methods for introducing genes into a cell and expressing them are known in the art. In the case of an expression vector, the vector can easily be introduced into a host cell, such as a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector can be transferred into the host cell using a physical, chemical or biological method.

[000316] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for obtaining cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In embodiments, the method of introducing the polynucleotide into the host cell is calcium phosphate transfection or polyethyleneimine (PEI) transfection.

[000317] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and in particular retroviral vectors, have become the most widely used method for inserting genes into mammals, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[000318] Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane vesicle).

[000319] When a viral delivery system is used, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. A nucleic acid associated with a lipid can be encapsulated in the aqueous interior of the liposome, embedded in the lipid bilayer of the liposome, attached to the liposome by a connecting molecule that is associated with both the liposome and the oligonucleotide embedded in the liposome, can form a complex with the liposome , may be dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, may be suspended in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Compositions associated with a lipid, lipid/DNA, or lipid/expression vector are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or in a "compressed" structure. They may also simply be interspersed in the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be natural or synthetic lipids. For example, lipids include fat droplets that naturally occur in the cytoplasm, as well as a class of compounds that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[000320] Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO, USA; dicetyl phosphate (“DCP”) can be obtained from K&K Laboratories (Plainview, NY, USA); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Alabama, USA). Lipid stock solutions in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term including a plurality of single and multilayer lipid carriers formed as a result of the production of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes have several lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo spontaneous rearrangement before forming closed structures and trapping water and dissolved analytes between lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, compositions are also contemplated whose structures in solution differ from the normal vesicular structure. For example, lipids can acquire a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also provided.

Non-viral delivery systems

[000321] In some instances, the polynucleotides encoding the cell labels described herein can also be introduced into T cells using non-viral delivery systems such as the "Sleeping Beauty (SB) transposon system", which refers to a synthetic DNA- transposon for introducing DNA sequences into vertebrate chromosomes. Some exemplary embodiments of the system are described, for example, in US Pat. In embodiments, the Sleeping Beauty transposon system may include an SB11 transposon system, an SB100X transposon system, or an SB110 transposon system.

[000322] DNA transposons move from one DNA site to another using a simple cut and paste method. Transposition is a precise process in which a specific segment of DNA is cut from one DNA molecule and moved to a different site in the same or a different DNA molecule or genome. Like other transposases of the Tc1/mariner type, the SB transposase inserts a transposon into a TA dinucleotide base pair in the recipient DNA sequence. The insertion site may be elsewhere on the same DNA molecule or on a different DNA molecule (or chromosome). There are approximately 200 million TA sites in the genomes of mammals, including humans. The TA insertion site is duplicated during transposon integration. This duplication of the TA sequence is a hallmark of transposition and has been used to elucidate the mechanism in some experiments. The transposase can be encoded either within the transposon, or the transposase can be supplied by another source, such as a DNA or mRNA source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are the most suitable as genetic tools because, once inserted, they cannot continue cutting and reinserting on their own. It seems possible to use SB transposons as non-viral vectors for introducing genes into vertebrate genomes and for gene therapy. In general terms, the Sleeping Beauty (SB) system (Hackett et al., Mol Ther 18:674-83, (2010)) has been adapted for the genetic modification of T cells (Cooper et al., Blood 105:1622-31, ( 2005)). This involved two steps: (i) electrotransfer of DNA plasmids expressing the SB transposon [i.e. chimeric antigen receptor (CAR) for redirecting T cell specificity (Jin et al., Gene THER 18:849-56, (2011); Kebriaei et al., Hum Gene THER 23:444-50, (2012)] and transposase SB, and (ii) growing and propagating T cells stably expressing integrated elements on original artificial antigen presenting cells (AaPCs) derived from the K562 cell line (also known as AaPCs (activating and promoting cells). According to one embodiment of the present of the invention, an SB transposon system includes a coding sequence encoding membrane-bound IL-15 and/or a chimeric antigen receptor Such systems are described, for example, in Singh et al., Cancer Res (8):68 (2008) April 15, 2008 and Maiti et al., J ImmunotHER 36(2): 112-123 (2013), incorporated herein by reference in its entirety In certain embodiments, a DNA plasmid expressing an SB transposon containing a CAR or TCR, and a cytokine and a a cell label is introduced by electroporation into the effector cell, which is then administered to the patient without further cultivation or propagation.

[000323] According to some embodiments of the present invention, a polynucleotide encoding a CAR or TCR, or a cytokine and one or more polypeptide cell tags in the modified effector cell described in this application, is encoded in one or more transposon vectors based on a DNA plasmid, and the transposase SB is encoded in a separate vector. In embodiments, the polypeptide cell tag is encoded in a DNA plasmid transposon vector, CAR or TCR, or the cytokine is encoded in a second DNA plasmid transposon vector and the SB transposase is encoded in a third DNA plasmid vector. In some embodiments of the present invention, the CAR or TCR and the cytokine and the polypeptide cell tag are encoded in the same transposon.

[000324] In some embodiments, the polypeptides described herein provide a protective mechanism by allowing infused CAR-T cells to be depleted by administration of FDA-approved antibodies or any antibody which recognizes a polypeptide construct to induce a cell depletion pathway. In some embodiments of the present invention, the HER1t variants described herein provide a protective mechanism by binding to administered cetuximab and causing cell depletion. In some embodiments of the present invention, CD20t variants also provide a safety mechanism by allowing infused CAR-T cells to be depleted by administration of FDA-approved rituximab therapy.

[000325] Regardless of the method used to introduce exogenous nucleic acids into the host cell, or otherwise expose the cell to an inhibitor of the present invention, various assays can be performed to confirm the presence of a recombinant DNA sequence in the host cell. Such assays include, for example, molecular assays well known to those skilled in the art such as Southern blot and Northern blot, RT-PCR and PCR; "biochemical" assays, such as detection of the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blot) or assays described in this application, to identify agents included in the scope of the present invention.

[000326] In embodiments, the modified effector cell described herein and other genetic elements are delivered to the cell using the SB11 transposon system, the SB100X transposon system, the SB110 transposon system, the piggyBac transposon system (see, e.g., Wilson et al, " PiggyBac Transposon-mediated Gene Transfer in Human Cells," Molecular THERapy 15:139-145 (2007), which is hereby incorporated by reference in its entirety) and/or the piggyBac transposon system (see, e.g., Mitra et al., "Functional "characterization of piggyBac from the bat Myotis lucifugus unveils an active mammalian DNA transposon," Proc. Natl. Acad. Sci USA 110:234-239 (2013). 7985739, 8227432, 9228180, U.S. Patent Publication No. 2011/0117072, Mates et al., Nat Genet, 41(6):753-61 (2009), doi: 10.1038/ng.343, Epub 2009 May 3, Gene THER. , 18(9):849-56 (2011). doi: 10.1038/gt.2011.40. Epub 2011 Mar 31 and in Ivies et al., Cell. 91(4):501-10, (1997), each of which is incorporated herein by reference in its entirety. Additional suitable non-viral systems may include integrating expression vectors, which may randomly integrate into the DNA of the host cell, or may contain a recombination site to allow for specific recombination between the expression vector and the chromosome of the host cell. Targeted integration of transgenes at predetermined genetic loci is a desirable goal for many applications. First, a first recombination site for a site-specific recombinase is inserted into the genomic site, either at random or at a predetermined location. The cells are then transfected with a plasmid carrying the gene or DNA of interest, the second recombination site, and the recombinase source (expression plasmid, RNA, protein, or virally expressed recombinase). Recombination between the first and second recombination sites results in integration of the plasmid DNA.

[000327] In such integrating expression vectors, endogenous expression control sequences from the chromosomes of the host cell can be used to express the target protein. In some embodiments of the present invention, targeted integration is stimulated by the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site. For example, targeted integration using the donor polynucleotides described herein can be achieved using conventional transfection techniques, such as techniques used to knock out or insert genes by homologous recombination. In other embodiments of the present invention, targeted integration is promoted by both having sequences on the donor polynucleotide that are homologous to sequences flanking the integration site, and by bringing the cells into contact with the donor polynucleotide in the presence of a site-specific recombinase. By site-specific recombinase, or simply recombinase, is meant a polypeptide that catalyzes conserved site-specific recombination between its compatible recombination sites. In the present application, a site-specific recombinase includes native polypeptides, as well as derivatives, variants and/or fragments that retain activity, and native polynucleotides, derivatives, variants and/or fragments that encode a recombinase that retains activity.

[000328] The present invention also provides a system for integrating heterologous genes into a host cell, said system comprising one or more gene expression cassettes. In some instances, the system comprises a first gene expression cassette containing a first polynucleotide encoding a first polypeptide construct. In other instances, the system may comprise a second gene expression cassette containing a second polynucleotide encoding a second polypeptide construct. In other cases, the system may contain a third gene expression cassette. According to one embodiment of the present invention, the system contains recombinant attachment sites; and a series of new recombinase; so that when said host cell is contacted with at least said first gene expression cassette in the presence of said serine recombinase, said heterologous genes are integrated into said host cell.

[000329] In some cases, the system further comprises a ligand; such that upon contact with said host cell in the presence of said ligand, said heterologous gene is expressed in said host cell. In one instance, the system also contains recombinant attachment sites. In some cases, one recombination attachment site is a recombination attachment site from the phage genome (attP) or a recombination attachment site from the bacterial genome (attB). In one instance, the host cell is a eukaryotic cell. In another case, the host cell is a human cell. In other cases, the host cell is a T cell or NK cell.

[000330] According to one embodiment of the present invention, the heterologous gene in the system described above contains CAR. In some embodiments, the CAR binds at least one of: CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2 .

[000331] According to another embodiment of the present invention, the system contains a heterologous gene containing a cytokine. In some cases, the cytokine contains at least one of: IL-15, IL-2, IL-12, IL-21, as well as a hybrid of IL-15 and IL-15Rα. According to some variants of implementation of the present invention, the system contains a heterologous gene containing at least one cell label described in this application. According to some variants of implementation of the present invention, the specified cell label contains at least one of: a HER1 polypeptide, a LNGFR polypeptide, a CD20 polypeptide, and a CD52 polypeptide. In some embodiments of the present invention, mbIL-15 is encoded with a cell tag. Examples of cell labels may include a truncated: HER1 polypeptide, LNGFR polypeptide, CD20 polypeptide, CD52 polypeptide, or any other suitable cell label for use in depletion or as a kill switch or enrichment marker.

[000332] According to additional variants of implementation of the present invention, the specified system is contained in one or more vectors. In one case, the system is contained in one vector. In one instance, the first gene expression cassette, the second gene expression cassette, and the recombinant attachment sites are contained in the same vector. In one instance, the first gene expression cassette, the second gene expression cassette, the third gene expression cassette, and the recombinant attachment sites are contained in the same vector. In another case, the serine recombinase is SF370. In other cases, the serine recombinase is in a separate vector.

[000333] The recombinases can be introduced into the target cell before, simultaneously with, or after the introduction of the targeting vector. The recombinase can be directly introduced into the cell as a protein, for example using liposomes, coated particles or microinjection. In another embodiment, the polynucleotide, either DNA or messenger RNA, encoding the recombinase can be introduced into the cell using a suitable expression vector. The vector targeting components described above can be used in the construction of expression cassettes containing sequences encoding the recombinase of interest. can be selectively induced or repressed).

[000334] Recombinases for use in the implementation of the present invention can be obtained recombinantly or purified as described previously. Polypeptides having the desired recombinase activity can be purified to the desired purity using methods known in the art for protein ammonium sulfate precipitation and purification, including, but not limited to, size fractionation, affinity chromatography, HPLC, ion exchange chromatography, heparin-agarose affinity chromatography (e.g. Thorpe & Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998.)

[000335] According to one embodiment of the present invention, recombinases can be introduced into eukaryotic cells that contain recombination sites of attachment and in which recombination is desired, using any suitable method. Methods for introducing functional proteins into cells, for example by microinjection or other means, are well known in the art. The introduction of a purified recombinase protein provides short-term availability of the protein and its function, which is often the preferred embodiment. In another embodiment, the gene encoding the recombinase may be included in an expression vector used to transform the cell, wherein the polynucleotide encoding the recombinase is operably linked to a promoter that mediates expression of the polynucleotide in a eukaryotic cell. The recombinase polypeptide can also be introduced into a eukaryotic cell by a messenger RNA that encodes the recombinase polypeptide. It is generally preferred that the recombinase be present only for the time necessary to insert the nucleic acid fragments into the gene to be modified. Thus, the lack of a consistent presence associated with most expression vectors is not expected to be disadvantageous. The recombinase gene may be introduced into the cell before, after, or simultaneously with the introduction of the exogenous polynucleotide of interest. According to one embodiment of the present invention, the recombinase gene is present in a vector that carries the polynucleotide to be inserted; the recombinase gene may even be included in the polynucleotide. In other embodiments of the present invention, the recombinase gene is introduced into a transgenic eukaryotic organism. Transgenic cells or animals can be generated that express the recombinase constitutively or under the control of cell-specific, tissue-specific, developmental stage-specific, organelle-specific promoters, or promoters induced or repressed by a small molecule. Recombinases can also be expressed as a fusion protein with other peptides, proteins, nuclear localization signal peptides, signal peptides, or organelle-specific signal peptides (eg, mitochondrial or chloroplast transit peptides to facilitate recombination in mitochondria or chloroplasts).

[000336] For example, the recombinase may be from the integrase or resolvas families. The integrase family of recombinases has over one hundred members and includes, for example, FLP, Cre, and lambda integrase. The integrase family, also called the tyrosine family or the lambda integrase family, uses the catalytic hydroxyl group of tyrosine to nucleophilically attack the phosphodiester bond of DNA. Typically, members of the tyrosine family first cut the DNA, which later forms a double strand break. Examples of integrases from the tyrosine family include Cre, FLP, SSV1, and lambda (A) integrase. In the resolvase family, also known as the serine recombinase family, a conserved serine residue forms a covalent bond to a DNA target site (Grindley, et al., (2006) Ann Rev Biochem 16:16).

[000337] According to one embodiment of the present invention, the recombinase is an isolated polynucleotide sequence containing a nucleic acid sequence that encodes a recombinase selected from the group consisting of SPβc2 recombinase, SF370.1 recombinase, Bxb1 recombinase, A118 recombinase, and φRv1 recombinase. Examples of serine recombinases are described in detail in US Pat. No. 9,034,652, incorporated herein by reference in its entirety.

[000338] According to one embodiment of the present invention, a site-specific recombination method includes providing a first recombination site and a second recombination site; bringing the first and second recombination sites into contact with a prokaryotic recombinase polypeptide, which leads to recombination between recombination sites, and the specified recombinase polypeptide can mediate recombination between the first and second recombination sites, while the first recombination site is attP or attB, the second recombination site is is attB or attP, and the recombinase is selected from the group consisting of Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase, and Mycobacterium smegmatis phage recombinase, provided that if the first recombination attachment site is attB , the second recombination attachment site is attP, and if the first recombination attachment site is attP, the second recombination attachment site is attB. In further embodiments, the introduction of a site-specific recombinase into a cell whose genome is to be modified is provided. One implementation variant relates to a method of obtaining site-specific recombination in a eukaryotic cell, including providing a eukaryotic cell that contains a first recombination attachment site and a second recombination attachment site; bringing the first and second recombination attachment sites into contact with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein said recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, wherein the first recombination attachment site is a recombination attachment site from a phage genome (attP) or a recombination attachment site from a bacterial genome (attB), the second recombination attachment site is attB or attP, and the recombinase is selected from the group consisting of Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase and Mycobacterium smegmatis phage recombinase, provided that if the first recombination attachment site is attB, the second recombination attachment site is attP, and if the first recombination attachment site is attP, the second recombination attachment site is attB. According to one embodiment of the present invention, the recombinase is selected from the group consisting of A118 recombinase, SF370.1 recombinase, SPβc2 recombinase, φRv1 recombinase, and Bxb1 recombinase. According to one embodiment of the present invention, recombination leads to integration.

Sources of immune effector cells

[000339] In certain aspects, the embodiments described herein include methods for generating and/or propagating antigen-specific retargeted immune effector cells (e.g., T cells, NK cells, or NK T cells), which include transfecting the cells with an expression vector, containing a DNA (or RNA) construct encoding a polypeptide construct, then optionally stimulating the cells using feeder cells, a recombinant antigen, or an antibody to the receptor to induce cell proliferation. In certain aspects, a cell (or population of cells) modified to express a CAR or TCR is a stem cell, an induced pluripotent stem cell (iPSC), an immune effector cell, or a precursor of these cells.

[000340] Sources of immune effector cells can include both allogeneic and autologous sources. In some cases, immune effector cells can be differentiated from stem cells or induced pluripotent stem cells (iPSCs). Thus, the cell to be modified in accordance with embodiments may be isolated from cord blood, peripheral blood, human embryonic stem cells, or iPSCs. For example, allogeneic T cells can be modified to include a chimeric antigen receptor (and optionally without a functional TCR). In some aspects, the immune effector cells are primary human T cells, such as T cells derived from human peripheral blood mononuclear cells (PBMCs). PBMCs can be collected from peripheral blood or after stimulation with G-CSF (granulocyte colony stimulating factor) from bone marrow or cord blood. Following transfection or transduction (eg, using a CAR expression construct), the cells may be immediately infused or may be cryopreserved. In certain aspects, after transfection, cells can be expanded for days, weeks, or months ex vivo as a general population about 1, 2, 3, 4, 5 days or more after gene transfer to cells. In a further aspect, after transfection, the transfectants are cloned, and a clone showing the presence of a separate integrated or episomal maintained expression cassette or plasmid, as well as expression of the chimeric antigen receptor, is propagated ex vivo. The clone selected for propagation demonstrates the ability to specifically recognize and lyse target cells expressing the antigen. Recombinant T cells can be expanded by stimulation with IL-2 or other cytokines that bind a common gamma chain (eg, IL-7, IL-12, IL-15, IL-21, and others). Recombinant T cells can be expanded by stimulation using artificial antigen presenting cells. Recombinant T cells can be propagated on an artificial antigen presenting cell or with an antibody such as OCTP that crosslinks CD3 on the surface of the T cells. Subsets of recombinant T cells can be further selected using magnetic bead isolation techniques and/or fluorescence activated cell sorting technology, and can then be cultured with AaPC. In a further aspect, the genetically modified cells may be cryopreserved.

[000341] T cells can also be obtained from a number of sources, including peripheral blood, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumor (tumor-infiltrating lymphocytes) . In certain embodiments of the present invention, any number of T cell lines available in the art may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood taken from a subject using any number of techniques known to one of skill in the art, such as Ficoll® separation. In embodiments, cells are obtained from the individual's circulating blood by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment of the present invention, the cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or media for subsequent processing steps. According to one implementation variant of the present invention, the cells are washed with phosphate-buffered saline (PBS). In another embodiment, the wash solution does not contain calcium and may not contain magnesium, or may not contain many or even all of the divalent cations. The initial stages of activation in the absence of calcium lead to enhanced activation. Those skilled in the art will readily appreciate that the washing step can be performed using methods known to those skilled in the art, such as using a semi-automatic flow through centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the instructions. manufacturer. After washing, the cells can be resuspended in various biocompatible buffers such as, for example, Ca ²⁺ free, Mg ²⁺ free PBS, PlasmaLyte A, or other buffered or unbuffered saline. Alternatively, unwanted components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[000342] According to another embodiment of the present invention, T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, for example, using PERCOLL® gradient centrifugation or flow elution centrifugation. A specific subset of T cells, such as CD3 ⁺ , CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and CD45RO ⁺ T cells, can be further isolated using positive or negative selection techniques. According to another embodiment of the present invention, CD14 ⁺ cells are depleted from a population of T cells. For example, according to one embodiment of the present invention, T cells are isolated by incubation with anti-CD3/CD28 antibody conjugated beads (i.e. 3×28), such as DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient for positive selection of target T cells. According to one embodiment of the present invention, the time period is about 30 minutes. According to a further implementation, the time period is from 30 minutes to 36 hours or more, including all integer values in between. According to a further embodiment of the present invention, the time period is at least 1, 2, 3, 4, 5, or 6 hours. According to another embodiment of the present invention, the time period is from 10 to 24 hours. According to one embodiment of the present invention, the incubation period is 24 hours. For the isolation of T cells from patients with leukemia, the use of a longer incubation time, such as 24 hours, may increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor infiltrating lymphocytes (IOLs) from tumor tissue or in immunosuppressed individuals. In addition, the use of longer periods of incubation time can increase the efficiency of capture CD8 ⁺ T cells. Thus, simply reducing or increasing the time that T cells can bind to CD3/CD28 granules, and/or increasing or decreasing the ratio of granules to T cells (as described later in this application), may allow preferential selection of the T subset. -cells by the presence of any feature or its absence at the beginning of cultivation or at other times during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, T cell subpopulations can be preferentially selected for the presence or absence of any trait at the start of culture or at other desired time points. . One skilled in the art will appreciate that multiple selection rounds can also be applied to the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and propagation process. "Unselected" cells may also be subjected to additional rounds of selection.

[000343] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry, which uses a set of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail typically contains antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments of the present invention, it may be desirable to enrich or positively select for regulatory T cells that normally express CD4 ⁺ , CD25 ⁺ , CD62Lhi, GITR ⁺ and FoxP3 ⁺ . Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25 antibody-conjugated beads or other similar selection method.

[000344] In order to isolate the target population of cells by positive or negative selection, the concentration of cells and surface (eg, particles such as beads) can be varied. According to certain embodiments of the present invention, it may be desirable to significantly reduce the volume in which the beads and cells are mixed (ie, increase the concentration of cells) in order to maximize contact between cells and beads. For example, according to one embodiment of the present invention, a concentration of 2 billion cells/mL is used. In one embodiment of the present invention, a concentration of 1 billion cells/mL is used. According to a further embodiment of the present invention, more than 100 million cells/ml are used. According to a further embodiment of the present invention, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. According to another embodiment of the present invention, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. According to additional embodiments of the present invention, concentrations of 125 or 150 million cells/ml can be used. The use of high concentrations can lead to increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples that contain many tumor cells (eg, leukemic blood, tumor tissue etc.). Such populations of cells may be of therapeutic value and it may be desirable to obtain them. For example, the use of a high concentration of cells allows more efficient selection of CD8 ⁺ T cells, which usually have a weaker expression of CD28.

[000345] In a related embodiment, the use of lower cell concentrations may be desirable. By greatly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This allows selection of cells that express large amounts of target antigens to be associated with the particles. For example, CD4 ⁺ T cells express higher levels of CD28 and are captured more efficiently than CD8 ⁺ T cells at dilute concentrations. According to one implementation variant of the present invention, the applied concentration of cells is 5×10 ⁶ /ml. In other embodiments of the present invention, the applied concentration may be from about 1×10 ⁵ /ml to 1×10 ⁶ /ml, including any integer value in between.

[000346] According to other variants of implementation of the present invention, cells can be incubated on a rotating device for various periods of time at different speeds, either at 2-10°C or at room temperature.

[000347] T cells for stimulation can also be frozen after the washing step. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be suitable in this case, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% PlasmaLyte A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% whey human albumin and 7.5% DMSO, or other suitable cell freezing media containing e.g. Hespan and PlasmaLyte A, the cells are then frozen to -80°C at 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank . You can use other methods of controlled freezing, as well as uncontrolled freezing directly at -20°C or in liquid nitrogen.

[000348] According to certain embodiments of the present invention, cryopreserved cells are thawed and washed as described herein and left alone for one hour at room temperature before being activated using the methods of the present invention.

[000349] The present invention also contemplates collecting blood or apheresis product samples from a subject during a period of time prior to when the expanded cells described herein may be required. In this regard, the source of cells to be expanded can be collected at any desired time, and target cells, such as T cells, can be isolated and frozen for subsequent use in T cell therapy for any number of diseases or conditions, for which T-cell therapy may be beneficial, such as those described in this application. In one embodiment of the present invention, a blood sample or apheresis product is collected from a generally healthy subject. In certain embodiments of the present invention, a blood sample or apheresis product is collected from a generally healthy subject who is at risk for developing a disease but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments of the present invention, T cells can be expanded, frozen, and used at a later time. In certain embodiments of the present invention, samples are taken from a patient shortly after the diagnosis of the specific disease described herein, but prior to any treatment. In a further embodiment of the present invention, cells are isolated from a blood sample or apheresis product from a subject prior to initiation of any number of appropriate treatments, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and radiation. These drugs inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit the p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, (1991); Henderson et al. ., Immun 73:316-321, (1991); Bierer et al., Curr. Opin. Immun 5:763-773, (1993)). In a further embodiment of the present invention, cells are isolated from a patient and frozen for later use in conjunction with (e.g., before, concurrently, or after) bone marrow or stem cell transplantation, T cell ablative therapy using any chemotherapeutic agents such as fludarabine, topical external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment of the present invention, the cells are isolated in advance and can be frozen for later use for treatment following B cell ablative therapy, eg, agents that react with CD20, eg, Rituxan.

[000350] According to an additional embodiment of the present invention, T cells are obtained from the patient immediately after treatment. In this regard, it has been found that after certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during a period when patients would normally recover from treatment, the quality of the resulting T cells may be optimal. or improved in terms of their ability to propagate under ex vivo conditions. Similarly, after ex vivo manipulation using the methods described in this application, these cells may be in a preferred state for increased engraftment and reproduction under in vivo conditions. Thus, the present invention provides for the collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. In addition, according to certain embodiments of the present invention, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject that stimulates repopulation, recycling, regeneration and/or multiplication of specific cell types, in particular, within a certain time window after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Activation and reproduction of T cells

[000351] According to certain embodiments of the present invention, T cells containing the polynucleotides and polypeptides described in this application can optionally be activated and propagated, usually using the methods described, for example, in US patent No. 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041; and U.S. Patent Application Publication No. 20060121005.

[000352] "Adoptive T cell transfer" refers to ex vivo isolation and expansion of tumor-specific T cells to produce a greater number of T cells than would be obtained by vaccination or the patient's natural tumor response alone. Tumor-specific T cells are then infused into cancer patients to give their immune system the ability to suppress the remaining tumor with T cells that can attack and destroy the cancer. There are many forms of adoptive T cell therapy used to treat cancer; culturing tumor-infiltrating lymphocytes or IOLs, isolating and expanding one particular T cell or clone, and even using T cells that have been modified to effectively recognize and attack tumors.

[000353] In some instances, the T cells described herein are propagated by contact with a surface to which is attached an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or with an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (for example, bryostatin) in combination with a calcium ionophore. To costimulate a helper molecule on the surface of T cells, a ligand is used that binds the helper molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate the proliferation of both CD4 ⁺ T cells and CD8 ⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody are used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), other methods well known in the art can also be used (Berg et al., Transplant Proc. 30(8):3975-3977 , (1998) Haanen et al., J. Exp. Med. 190(9): 13191328, (1999) Garland et al., J. Immunol Meth. 227(1-2):53-63, (1999 )).

[000354] According to certain embodiments of the present invention, the primary stimulatory signal and the co-stimulatory signal for T cells can be provided using various protocols. For example, the agents providing each signal may be in solution or may be associated with the surface. If agents are surface bound, they may be bound to the same surface (ie, in "cis" form) or to separate surfaces (ie, in "trans" form). According to another option, one agent may be associated with the surface, and the other agent may be in solution. In one embodiment of the present invention, the agent providing the costimulatory signal is associated with the cell surface, and the agent providing the primary activating signal is in solution or associated with the surface. In certain embodiments of the present invention, both agents may be in solution. According to another embodiment of the present invention, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing Fc receptors, or an antibody or other binding agent that will bind to the agents. In this regard, see, for example, US Patent Application Publication Nos. 20040101519 and 20060034810 which describe artificial antigen presenting cells (iAPCs) which are contemplated for use in T cell activation and expansion in the present invention.

[000355] According to one embodiment of the present invention, the two agents are immobilized on the beads, or on the same bead, i. "cis", or on separate granules, i.e. "trance". For example, an agent providing a primary activating signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and an agent providing a costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment of the present invention, each antibody bound to the CD4 ⁺ T cell expansion and T cell expansion beads is used in a 1:1 ratio. In certain aspects of the present invention, a ratio of bead-bound anti-CD3:CD28 antibodies is used such that there is an increase in T cell expansion compared to that observed when using a 1:1 ratio. In one particular embodiment, there is an increase from about 1 to about 3 times the expansion observed when using a 1:1 ratio. According to one implementation variant of the present invention, the ratio of antibodies to CD3:CD28 associated with granules is in the range from 100:1 to 1:100 and all integer values between them. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i. e. the CD3:CD28 ratio is less than one. In certain embodiments of the present invention, the ratio of anti-CD28 antibody to bead-bound anti-CD3 antibody is greater than 2:1. In one specific embodiment, a 1:100 ratio of anti-CD3:CD28 antibodies associated with the beads is used. In another embodiment, a 1:75 ratio of anti-CD3:CD28 antibodies associated with the beads is used. According to a further embodiment of the present invention, a 1:50 ratio of anti-CD3:CD28 antibodies associated with the beads is used. According to another embodiment of the present invention, a 1:30 ratio of anti-CD3:CD28 antibodies associated with beads is used. Embodiments use a 1:10 ratio of anti-CD3:CD28 antibodies associated with the beads. According to another embodiment of the present invention, a 1:3 ratio of anti-CD3:CD28 antibodies associated with beads is used. According to another embodiment of the present invention, a 3:1 ratio of anti-CD3:CD28 antibodies associated with beads is used.

[000356] To stimulate T cells or other target cells, particle to cell ratios of 1:500 to 500:1 and any integer values in between can be used. Those of ordinary skill in the art will readily appreciate that the ratio of particles to cells may depend on the size of the particles relative to the target cell. For example, small granules may bind only a few cells, while large granules may bind many cells. According to certain embodiments of the present invention, the ratio of cells to particles is in the range from 1:100 to 100:1 and any integer values in between, and according to additional embodiments of the present invention, the ratio is from 1:9 to 9:1 and any integer values between them, which can be used to stimulate T-cells. The ratio of anti-CD3 antibody and anti-CD28 antibody bound particles to T cells that results in T cell stimulation can vary as above, but specific values include 1:100, 1:50, 1:40, 1 :30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1 , 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, with one ratio being at least 1:1 particles per T cell. In one embodiment of the present invention, a particle to cell ratio of 1:1 or less is used. According to one particular embodiment of the present invention, the particle:cell ratio is 1:5. According to additional variants of implementation of the present invention, the ratio of particles and cells may vary depending on the day of stimulation. For example, according to one embodiment of the present invention, the ratio of particles to cells is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or a day thereafter for up to 10 days at final ratios from 1:1 up to 1:10 (based on the number of cells on the day of addition). In one particular embodiment, the particle to cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on the third and fifth days of stimulation. According to another embodiment of the present invention, the particles are added daily or every other day to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. According to another embodiment of the present invention, the particle to cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. According to another embodiment of the present invention, the particles are added daily or every other day to a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. One skilled in the art will appreciate that many other ratios may be suitable for use in the present invention. In particular, the ratios will vary with particle size as well as cell size and type.

[000357] In further embodiments of the present invention, cells such as T cells are combined with agent-coated beads, whereafter the beads and cells are separated, and then the cells are cultured. In an alternative embodiment, the agent-coated beads and cells are not separated but co-cultured prior to culturing. According to a further embodiment of the present invention, the beads and cells are first concentrated by applying a force, such as a magnetic force, this results in increased ligation of cell surface markers, which induces stimulation of the cells.

[000358] For example, cell surface proteins can be ligated to contact the paramagnetic beads to which the anti-CD3 antibody and anti-CD28 antibody (3x28 beads) are attached to T cells. In one embodiment of the present invention, cells (e.g., 10 ⁴ -10 ⁹ T cells) and beads (e.g., DYNABEADS® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio or MACS® MicroBeads from Miltenyi Biotec) are combined in buffer, such as PBS (without divalent cations such as calcium and magnesium). Also, those of ordinary skill in the art will readily be able to determine any concentration of cells that can be used. For example, the target cell may be very rare in the sample and may comprise only 0.01% of the sample, or the entire sample (ie 100%) may be composed of target cells of interest. Accordingly, any number of cells is included within the scope of the present invention. According to certain embodiments of the present invention, it may be desirable to significantly reduce the volume in which the particles and cells are mixed (ie, increase the concentration of cells) in order to maximize the contact of cells and particles. For example, according to one embodiment of the present invention, a concentration of about 2 billion cells/mL is used. In another embodiment, more than 100 million cells/ml are used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. According to another additional embodiment of the present invention, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. According to additional embodiments of the present invention, concentrations of 125 or 150 million cells/ml can be used. The use of high concentrations can lead to increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may be of therapeutic value, and their production is desirable in certain embodiments of the present invention. For example, the use of a high concentration of cells allows more efficient selection of CD8 ⁺ T cells, which usually have a weaker expression of CD28.

[000359] According to one implementation variant of the present invention, the mixture can be cultured from several hours (about 3 hours) to about 14 days, or any integer number of hours in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the present invention, the beads and T cells are cultured together for about eight days. According to another embodiment of the present invention, the beads and T cells are cultured together for 2-3 days. Multiple cycles of stimulation may also be desirable, so T cell culture time may be 60 days or more. Conditions suitable for culturing T cells include suitable media (eg, minimal culture media or RPMI 1640 or X-vivo 15 (Lonza) media) that may contain factors necessary for proliferation and viability, including serum (eg, fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF -alpha or any other cell growth additive known to the person skilled in the art. Other cell culture additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate and vitamins, either serum-free or supplemented with an appropriate amount serum (or plasma) or a certain set of hormones and / or an amount of cytokine (s) sufficient for the growth and reproduction of T cells. Antibiotics, such as penicillin and streptomycin, are only included in experimental cultures, not in cell cultures to be administered to a subject by infusion. The target cells are maintained under conditions necessary to support proliferation, eg at a suitable temperature (eg 37° C.) and atmosphere (eg air plus 5% CO ₂ ).

[000360] T cells that have been stimulated for different durations may exhibit different characteristics. For example, typical apheresis-derived blood or peripheral blood mononuclear cell products contain a population of helper T cells (TH, CD4 ⁺ ) that is larger than a population of cytotoxic or suppressor T cells (TC, CD8 ⁺ ). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors results in a T cell population that consists predominantly of TH cells up to about 8-9 days, while after about 8-9 days the T cell population contains an increasing population of TC cells. Accordingly, depending on the purpose of treatment, administration to a subject by infusion of a population of T cells containing predominantly TH cells may be preferred. Similarly, if an antigen-specific subset of TC cells has been isolated, expansion of that subset may be more favorable.

[000361] In addition, in addition to the CD4 and CD8 markers, other phenotypic markers vary significantly, but largely reproducibly, during the process of cell reproduction. Thus, this reproducibility allows the activated T cell product to be tailored for specific purposes.

[000362] In some cases, immune effector cells in embodiments (eg, T cells) are co-cultured with proliferating and activating cells (AaPCs) to facilitate cell expansion. AaRS may also be referred to as artificial antigen presenting cells (iAPCs). For example, antigen presenting cells (APCs) can be used in the preparation of therapeutic compositions and cell therapy products, according to embodiments. In one aspect, the AaPCs may be genetically modified K562 cells. For general guidance on the preparation and use of antigen presenting systems, see, for example, US Pat. Nos. 6,225,042; 6,355,479; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. WO2007/103009, each of which is incorporated by reference. According to another additional aspect of the embodiments, culturing the genetically modified CAR cells includes culturing the genetically modified CAR cells in the presence of dendritic cells or proliferation-activating and proliferating cells (AaPCs) that stimulate the proliferation of CAR-expressing immune effector cells. In other additional aspects, the AaPCs comprise a CAR-binding antibody, or fragment thereof, expressed on the surface of the AaPCs. AaRS may contain additional molecules that activate or co-stimulate T cells in some cases. Additional molecules may contain, in some cases, membrane-bound Cy cytokines. In other additional aspects, the AaPCs are inactivated or irradiated or tested to determine the presence and confirm the absence of infectious materials. According to other additional aspects, culturing the transgenic CAR cells in the presence of AaPC includes culturing the transgenic CAR cells in a medium containing soluble cytokines such as IL-15, IL-21 and/or IL-2. Cells can be cultured in a ratio of about 10:1 to about 1:10; from about 3:1 to about 1:5; from about 1:1 to about 1:3 (ratio of effector cells and AaPC); or in any range of ratios that can be obtained from the indicated values. For example, a co-culture of T cells and AaPC may be in a ratio of about 1:1, about 1:2, or about 1:3.

[000363] In one aspect, AaPCs can express CD137L. In some aspects, AaPCs can additionally express the antigen targeted by the CAR cell. In other aspects, AaPCs can additionally express CD19, CD64, CD86, or mIL15. In certain aspects, AaPCs can express at least one anti-CD3 antibody clone, such as, for example, OKT3 and/or UCHT1. In one aspect, AaPCs can be treated (eg, irradiated or treated with mitomycin C) to eliminate their ability to reproduce. In one aspect, the AaPCs could be tested to determine the presence or absence of infectious materials. Methods for producing such AaPCs are known in the art. In one aspect, culturing a population of CAR-modified T cells with AaPC may include culturing the cells at a ratio of about 10:1 to about 1:10; from about 3:1 to about 1:5; from about 1:1 to about 1:3 (ratio of T cells and AaPC); or in any range of ratios that can be obtained from the specified values. For example, a co-culture of T cells and AaPC may be in a ratio of about 1:1, about 1:2, or about 1:3. In one aspect, the culturing step may further include culturing with an aminobisphosphonate (eg, zoledronic acid).

[000364] In a further aspect, the CAR-T cell population is cultured and/or stimulated for no more than 7, 14, 21, 28, 35, 42 days, 49, 56, 63, or 70 days. In some embodiments of the present invention, a population of CAR-T cells is cultured and/or stimulated for at least 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more days. In some embodiments, the CAR-T cell population is cultured and/or stimulated for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days. In some embodiments, the CAR-T cell population is cultured and/or stimulated for at least 7, 14, 21, 28, 35, 42, 49, 56, 63 or more days. According to other embodiments of the present invention, stimulation comprises co-culture of CAR-T cells with AaPC to stimulate the expansion of CAR-positive T cells. In another aspect, a population of genetically modified CAR cells is stimulated with no more than: 1X stimulation, 2X stimulations, 3X stimulations, 4X stimulations, 5X stimulations, 5X stimulations, 6X stimulations, 7X stimulations, 8X stimulations, 9X stimulations, or 10X stimulations. In some cases, genetically modified cells are not cultured ex vivo in the presence of AaPC. In some specific cases, the method of an embodiment further comprises enriching the cell population with CAR-expressing immune effector cells (eg, T cells) after the transfection and/or culture step. Enrichment may include fluorescence activated cell sorting (FACS) and sorting of cells expressing CAR. In a further aspect, sorting to obtain CAR-expressing cells involves the use of a CAR-binding antibody. Enrichment may also include depletion of CD56 ⁺ cells. According to yet another additional aspect of the embodiment, the method further comprises cryopreserving a sample of the population of genetically modified CAR cells.

[000365] In some cases, AaRS are incubated with a peptide of optimal length, which allows the peptide to bind directly to the MHC molecule without additional processing. In another embodiment, the cells may express the antigen of interest (ie, in the case of MHC-independent antigen recognition). In addition, in some cases, APCs can express an antibody that binds either to a particular CAR polypeptide or to CAR polypeptides in general (e.g., a universal activating and proliferating cell (uAPC). Such methods are disclosed in WO/2014/190273, which includes to the present application by reference.In addition to the peptide-MHC molecules or antigens of interest, AaPC systems may also contain at least one exogenous accessory molecule.Any suitable number and combination of accessory molecules can be used.The accessory molecule can be selected from the accessory molecules , such as co-stimulatory molecules and adhesion molecules Exemplary co-stimulatory molecules include CD70 and B7.1 (B7.1 was formerly known as B7 and also known as CD80), which, among other things, bind to CD28 and/or CTLA-4 molecules on surface of T cells, thereby affecting, for example, T cell proliferation, Th1 differentiation, short term survival of T cells, and secretion of cytokines such as interleukin (IL)-2. Adhesion molecules can include carbohydrate-binding glycoproteins such as selectins, transmembrane-binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which stimulate , for example, cell-to-cell or cell-to-matrix contacts. Exemplary adhesion molecules include LFA-3 and ICAM such as ICAM-1. Methods, methods, and reagents suitable for selecting, cloning, generating, and expressing exemplary accessory molecules, including co-stimulatory molecules and adhesion molecules, are given, for example, in US Pat.

[000366] Cells selected for conversion to AaPC preferably have defects in intracellular antigen processing, intracellular peptide transport, and/or intracellular loading of an MHC class 1 or class II molecule with a peptide, or are poikilothermic (i.e., less temperature sensitive than lines mammalian cells), or have both defects and poikilothermic properties. Preferably, cells selected for conversion to AaPC are also unable to express at least one endogenous partner (e.g., an endogenous MHC class 1 or class II molecule and/or endogenous accessory molecules as described above) of an exogenous MHC class 1 or class II molecule, and components of auxiliary molecules that are injected into cells. In addition, AaPCs preferably retain the defects and poikilothermic properties that the cells had before they were modified to produce AaPCs. Exemplary AaPCs are or are derived from a cell line deficient in an antigen processing associated transporter (TAP), such as an insect cell line. An exemplary poikilothermic insect cell line is a Drosophila cell line such as the Schneider 2 cell line (see, for example, Schneider 1972). Illustrative methods for obtaining, growing and culturing Schneider 2 cells are presented in US patent Nos. 6225042, 6355479 and 6362001.

[000367] According to one embodiment of the present invention, AaPC is also subjected to a freeze-thaw cycle. In an exemplary freeze-thaw cycle, AaPC can be frozen by bringing a suitable vessel containing AaPC into contact with an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or similar low temperature material such that rapid freezing occurs. The frozen APCs are then thawed either by removing the AaPC from the low temperature material and subjecting to ambient room temperature conditions, or by using a facilitated thaw process in which a moderately warm water bath or warm hand is used to promote shorter thaw times. In addition, AaPC can be frozen and stored for an extended period of time before being thawed. Frozen AaPCs can also be thawed and then lyophilized before further use. Preferably, preservatives that can adversely affect freeze-thaw procedures, such as dimethyl sulfoxide (DMSO), polyethylene glycols (PEGs) and other preservatives, are absent from media containing AaPC that are subjected to a freeze-thaw cycle, or are substantially removed, for example, by transferring AaPC to media that are substantially free of such preservatives.

[000368] According to further embodiments of the present invention, the xenogeneic nucleic acid and the nucleic acid endogenous to AaPC can be inactivated by crosslinking such that, after inactivation, there is substantially no cell expansion, replication, or expression of the nucleic acid. According to one embodiment of the present invention, AaRS are inactivated at a point in time after expression of exogenous MHC and accessory molecules, presentation of such molecules on the surface of AaRS, and loading of the presented MHC molecules with the selected peptide or peptides. Accordingly, such AaPCs, inactivated and loaded with the selected peptide, despite being essentially devoid of the ability to proliferate or replicate, retain the function of presenting the selected peptide. Preferably, cross-linking also produces AaPCs that are substantially free of contaminating microorganisms such as bacteria and viruses without significantly reducing the antigen-presenting cellular function of AaPCs. Thus, cross-linking supports the important functions of AaPC while helping to reduce safety concerns for a cell therapy product developed using AaPC. Methods related to crosslinking and AaPC are described, for example, in US Patent Application Publication No. 20090017000, which is incorporated herein by reference.

[000369] According to certain embodiments of the present invention, a modified antigen presenting cell (APC) is further provided. Such cells can be used, for example, as described above, to propagate immune effector cells under ex vivo conditions. In additional aspects, the modified APCs can be administered to a patient as such, which stimulates the proliferation of immune effector cells in vivo. Modified APCs according to embodiments can be used as such as a therapeutic agent. According to other embodiments of the present invention, modified APCs can be used as a therapeutic agent that can stimulate the activation of endogenous immune effector cells specific for the target antigen and/or increase the activity or persistence of adaptively transferred immune effector cells specific for the target antigen. .

[000370] As used herein, the term "modified APC" refers to a cell(s) that contains(s) at least a first transgene, said first transgene encoding HLA. Such modified APCs may further comprise a second transgene for expressing the antigen such that the antigen is presented on the surface of the APC in association with HLA. In some aspects, the modified APC may be a cell type that presents antigens (eg, a dendritic cell). In additional aspects, the modified APC can be derived from a cell type that does not normally present antigens, such as a T cell or T cell progenitor (referred to as "T-APC"). Thus, in some aspects, the modified APC according to embodiments comprises a first transgene encoding a target antigen and a second transgene encoding a human leukocyte antigen (HLA), such that HLA is expressed on the surface of the modified APC in complex with the epitope of the target antigen. In certain aspects, the HLA expressed in the modified APC is HLA-A2.

[000371] In some aspects, the modified APC according to embodiments may further comprise at least a third transgene encoding a costimulatory molecule. The costimulatory molecule may be a costimulatory cytokine, which may be a membrane-bound cytokine Cy. In certain aspects, the costimulatory cytokine is IL-15, such as membrane-bound IL-15. In some additional aspects, the modified APC may contain an edited (or deleted) gene. For example, an inhibitory gene such as PD-1, LIM-3, CTLA-4, or TCR can be edited to reduce or eliminate gene expression. The modified APC according to embodiments may further comprise a transgene encoding any target antigen of interest. For example, the target antigen may be an infectious disease antigen or a tumor associated antigen (TAA).

Place of medical care

[000372] In one embodiment of the present invention, the immune effector cells described herein are modified at the point of care. In some instances, the point of care is in a hospital or facility (eg, medical facility) in the vicinity of the subject in need of treatment. The subject is undergoing apheresis and peripheral blood mononuclear cells (PBMCs) or a subset of PBMCs can be enriched, for example, by elutriation or ficoll separation. Enriched PBMCs or a subset of PBMCs can be cryopreserved in any suitable cryopreservation solution prior to further processing. In one case, the elutriation process is performed using a buffer solution containing human serum albumin. Immune effector cells, such as T cells, can be isolated using the selection methods described in this application. In one instance, the T cell selection method comprises beads specific for CD3 and CD8 on T cells. In one instance, the beads may be paramagnetic beads. The harvested immune effector cells can be cryopreserved in any suitable cryopreservation solution prior to modification. Immune effector cells can be thawed up to 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours before infusion. Prior to modification, thawed cells may be placed in a cell culture buffer, such as cell culture buffer (e.g., RPMI) supplemented with fetal bovine serum (FBS), or may be placed in a buffer that contains cytokines such as IL-2 and IL-21. In another aspect, the harvested immune effector cells can be immediately modified without the need for cryopreservation.

[000373] In some instances, immune effector cells are modified by modifying/introducing a chimeric receptor, one or more of the cell labels described herein, and/or a cytokine into immune effector cells, which are then rapidly infused into the subject. In some cases, sources of immune effector cells may include both allogeneic and autologous sources. In one instance, the immune effector cells may be T cells or NK cells. In one instance, the chimeric receptor may be the CD19 CAR. Alternatively, the chimeric receptor may be CD33CAR. In an additional case, the chimeric receptor may be a MUC16 CAR. Alternatively, the cytokine may be mbIL-15. In one case, mbIL-15 has the sequence of SEQ ID NO: 178, or a variant or fragment thereof. In one case, the cell label may have the sequence of SEQ ID NO: 57. In another case, the expression of mbIL-15 is modulated using expression systems with a ligand-induced gene switch described in this application. For example, a ligand such as veledimex can be delivered to a subject to modulate mbIL-15 expression. In another aspect, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg Veledimex is provided. In a further aspect, lower doses of veledimex are provided, eg 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg or 20 mg. According to one embodiment of the present invention, Veledimex is administered to the subject for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days prior to infusion of modified immune effector cells. According to a further embodiment of the present invention, Veledimex is administered to the subject after infusion of the modified immune effector cells about once every 12 hours, about once every 24 hours, about once every 36 hours, or about once every 48 hours, for an effective period of time. According to one implementation variant of the present invention, the effective period of time for the introduction of Veledimex is approximately: , 25, 26, 27, 28, 29, 30 days. According to other embodiments of the present invention, Veledimex may be re-administered after a rest period, after a drug interruption, or during a relapse in a subject.

[000374] In certain instances where an adverse effect on a subject is observed or when treatment is not required, a cell tag can be activated, such as with cetuximab, to conditionally ablate in vivo modified immune effector cells containing cell tags, such as tags based on the truncated epidermal growth factor receptor (HER1t variants) described in this application.

[000375] According to some embodiments of the present invention, such immune effector cells are modified with constructs introduced by electroporation. In one case, electroporation is performed using electroporators such as Nucleofector™ electroporators from Lonza. According to other embodiments of the present invention, the vector containing the above constructs is a non-viral or viral vector. In one case, the non-viral vector contains the Sleeping Beauty transposon-transposase system. In one case, immune effector cells are subjected to an irradiation using a specific sequence. For example, immune effector cells can be electroporated using one transposon and then the DNA encoding the transposase, followed by a second transposon. Alternatively, immune effector cells can be electroporated using all transposons and transposase simultaneously. Alternatively, immune effector cells can be electroporated using a transposase and then both transposons simultaneously or one transposon. In sequential electroporation, the immune effector cells may lie dormant for some period of time until the next stage of electroporation.

[000376] In some cases, the modified immune effector cells do not go through the expansion and activation phase. In some cases, modified immune effector cells do not go through an incubation or culture step (eg, ex vivo expansion). In certain cases, the modified immune effector cells are placed before infusion in a buffer that contains IL-2 and IL-21. In other instances, the modified immune effector cells are placed or dormant in cell culture buffer prior to infusion, such as cell culture buffer (eg, RPMI) supplemented with fetal bovine serum (FBS). Prior to infusion, the modified immune effector cells may be harvested, washed, and prepared in saline in preparation for infusion into a subject.

[000377] In one case, the subject was subjected to lymphatic depletion prior to infusion. In other cases, lymphatic depletion is not required and the modified immune effector cells are rapidly administered to the subject by infusion. Exemplary schemes of lymphatic depletion are given in tables 2 and 3 below:

[000378] In another case, the subject undergoes minimal lymphatic depletion. As used herein, minimal lymph depletion refers to an abbreviated lymph depletion protocol in which the subject may be infused for 1 day, 2 days, or 3 days after the lymph depletion regimen. In one case, an abbreviated lymphatic depletion protocol may include lower doses of fludarabine and/or cyclophosphamide. Alternatively, an abbreviated lymphatic depletion protocol may include a shortened lymphatic depletion period, such as 1 day or 2 days.

[000379] In one embodiment of the present invention, immune effector cells are modified by modifying/introducing a chimeric receptor and a cytokine into said immune effector cells, followed by rapid infusion into a subject. In other cases, immune effector cells are modified by modifying/introducing a chimeric receptor and a cytokine into said cells, followed by infusion into the subject for at least: 0, 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 hours. In other cases, the immune effector cells are modified by modifying/introducing a chimeric receptor and a cytokine into the immune effector cells, followed by infusion into the subject after 0 days, <1 day, <2 days, <3 days, <4 days, <5 days, <6 days or <7 days.

[000380] According to some embodiments of the present invention, an amount of modified effector cells is administered to a subject in need thereof, the amount being determined based on efficacy and possible induction of cytokine-associated toxicity. According to another embodiment of the present invention, the modified effector cells are CAR ⁺ and CD3 ⁺ cells. In some cases, the number of modified effector cells is from about 10 ⁴ to about 10 ⁹ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁴ to about 10 ⁵ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁵ to about 10 ⁶ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁶ to about 10 ⁷ modified effector cells/kg. In some cases, the number of modified effector cells is >10 ⁴ but ≤10 ⁵ modified effector cells/kg. In some cases, the number of modified effector cells is >10 ⁵ but ≤10 ⁶ modified effector cells/kg. In some cases, the number of modified effector cells is >10 ⁶ but ≤10 ⁷ modified effector cells/kg. According to one embodiment of the present invention, a lower dose of >10 ² but ≤10 ⁴ modified effector cells/kg can be administered by infusion.

[000381] According to one embodiment of the present invention, modified immune effector cells target cancer by local delivery directly to tumor tissue. For example, in ovarian cancer, modified immune effector cells can be delivered intraperitoneally (ip) to the abdomen or abdominal cavity. Such i.p. delivery can be done through a port or a pre-existing port placed for the delivery of chemotherapeutic drugs. Other methods of local delivery of modified immune effector cells may include infusion through a catheter into the resection cavity, ultrasound-guided intratumoral injection, hepatic artery infusion, or intrapleural delivery.

[000382] According to one embodiment of the present invention, a subject in need thereof may begin therapy with a first dose of modified immune effector cells delivered ip followed by a second dose of modified immune effector cells delivered iv. According to a further embodiment of the present invention, the second dose of modified immune effector cells may be followed by subsequent doses, which may be delivered iv or ip. According to one implementation variant of the present invention, the duration of the interval between the first and second or additional subsequent dose may be approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days. According to one implementation variant of the present invention, the duration of the interval between the first and second or additional subsequent dose may be approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 months. According to another embodiment of the present invention, the duration of the interval between the first and second or additional subsequent dose may be approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

[000383] According to another embodiment of the present invention, a catheter can be placed in a tumor or metastasis site for further administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 doses of modified immune effector cells. In some instances, doses of modified effector cells may be from about ¹⁰² to about ¹⁰⁹ modified effector cells/kg. In cases where toxicity is observed, doses of modified effector cells can be from about 10 ² to about 10 ⁵ modified effector cells/kg. In some cases, doses of modified effector cells may start at about 10 ² modified effector cells/kg, and subsequent doses can be increased to about: 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , or 10 ⁹ modified effector cells/kg .

[000384] According to other embodiments of the present invention, a method for promoting proliferation and/or survival of modified cells comprises obtaining a cell sample from a subject and transfecting cells from the cell sample with one or more polynucleotides that contain one or more transposons. In one embodiment of the present invention, the transposons encode a chimeric antigen receptor (CAR), a cytokine, one or more cell markers, and a transposase effective to integrate said one or more polynucleotides into the genome of said cells to provide a population of modified cells. In one embodiment of the present invention, the transposons encode a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, gene switch polypeptides for ligand-induced cytokine control, and a transposase effective to integrate said one or more polynucleotides into the genome of said cells to provide population of modified cells. In one embodiment, the gene switch polypeptides comprise i) a first gene switch polypeptide that contains a DNA binding domain hybridized to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that contains a transactivation domain hybridized to a second ligand binding domain. nuclear receptor. In some embodiments of the present invention, the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In one case, the subject does not require lymphatic depletion prior to administration of the modified cells. In one instance, a method for propagating modified cells in vivo includes obtaining a cell sample from a subject and transfecting the cells from the cell sample with one or more polynucleotides that contain one or more transposons. According to one embodiment of the present invention, a transposon(s) containing(s) a chimeric antigen receptor (CAR), a cytokine, one or more cell labels; and a transposase effective to integrate said one or more polynucleotides into the genome of said cells is introduced by electroporation into the cell sample to provide a population of modified cells. In a further embodiment, the transposons encode a chimeric antigen receptor (CAR), a cytokine, one or more cell markers, gene switch polypeptides for ligand-induced cytokine control, and a transposase effective to integrate said one or more polynucleotides into the genome of said cells to provide a population of modified cells. In one embodiment, the gene switch polypeptides comprise i) a first gene switch polypeptide that contains a DNA binding domain hybridized to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that contains a transactivation domain hybridized to a second ligand binding domain. nuclear receptor. In some embodiments of the present invention, the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. According to another embodiment of the present invention, one transposon may comprise a chimeric antigen receptor (CAR), a cytokine, one or more cell markers such as HER1t, or any of the variants described herein. In one case, the subject does not require lymphatic depletion prior to administration of the modified cells.

[000385] According to another embodiment of the present invention, a method for enhancing the in vivo stability of modified cells in a subject in need thereof comprises obtaining a cell sample from the subject and transfecting the cells from the cell sample with one or more polynucleotides that contain one or more transposons. According to one embodiment of the present invention, a transposon(s) containing(s) a chimeric antigen receptor (CAR), a cytokine, one or more cell labels; and a transposase effective to integrate said one or more polynucleotides into the genome of said cells is introduced by electroporation into the cell sample to provide a population of modified cells. According to another embodiment of the present invention, a single transposon may contain a chimeric antigen receptor (CAR), a cytokine, one or more cell markers such as HER1t, or any of the variants described herein. In some cases, one or more transposons encode a chimeric antigen receptor (CAR), a cytokine, one or more cell markers, gene switch polypeptides for ligand-induced cytokine control, and a transposase effective to integrate the DNA into the genome of said cells to provide a population of modified cells. In some instances, gene switch polypeptides comprise i) a first gene switch polypeptide that contains a DNA binding domain hybridized to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that contains a transactivation domain hybridized to a second nuclear receptor ligand binding domain, wherein said first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In one case, the subject does not require lymphatic depletion prior to administration of the modified cells.

[000386] According to another embodiment of the present invention, a method of treating a subject with a solid tumor comprises obtaining a cell sample from the subject, transfecting the sample cells with one or more polynucleotides that contain one or more transposons, and introducing the modified cell population to the subject. In one case, the subject does not require lymphatic depletion prior to administration of the modified cells. In some instances, one or more transposons encode a chimeric antigen receptor (CAR), a cytokine, one or more cell markers, and a transposase effective to integrate the DNA into the cell's genome. In some instances, one or more transposons encode a chimeric antigen receptor (CAR), a cytokine, one or more cell markers, gene switch polypeptides for ligand-induced cytokine control, and a transposase effective to integrate DNA into the cell genome. In some instances, gene switch polypeptides comprise: i) a first gene switch polypeptide that contains a DNA binding domain hybridized to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that contains a transactivation domain hybridized to a second nuclear receptor ligand binding domain wherein said first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In some cases, cells are transfected by electroporation. In some cases, polynucleotides encoding gene switch polypeptides are modulated with a promoter. In some cases, the promoter is a tissue-specific promoter or the EF1A promoter or a functional variant thereof. In some cases, the tissue-specific promoter contains a T cell response element or an NFAT response element. In some cases, the cytokine contains at least one of: IL-1, IL-2, IL-15, IL-12, IL-21, IL-15 hybrid, IL-15R, or IL-15 variant. In some cases, the cytokine is in a secreted form. In some cases, the cytokine is in a membrane bound form. In some instances, the cells are NK cells, NKT cells, T cells, or T cell progenitor cells. In some instances, the cells are administered to a subject (eg, by infusion of the modified cells into the subject). In some instances, the method further comprises administering an effective amount of a ligand (eg, Veledimex) to induce cytokine expression. In some cases, CAR is able to bind at least one of: CD19, CD33, BCMA, CD44, folate receptor α, 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, MAGE-A1, MUC-16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF -R2. In some cases, the transposase is a salmon-type Tc1-like transposase. In some cases, the transposase is an SB11 or SB100x transposase. In other cases, the transposase is PiggyBac. In some cases, the cell label contains at least one of HER1t1.

Indications for use

[000387] In some embodiments, this application discloses methods for introducing a modified effector cell encoding a polynucleotide described herein to a subject having a disorder, such as cancer or an infectious disease. In some cases, the cancer is a cancer associated with the expression of CD19, CD20, CD33, CD44, BCMA, CD123, EGFRvIII, folate receptor α, 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, MAGE-A1, h5T4, PSMA, TAG-72 or VEGF-R2.

[000388] In some embodiments, this application discloses methods of administering a polynucleotide, polypeptide, or modified effector cell encoding a polynucleotide described herein to a subject having cancer associated with CD19 overexpression. In some embodiments, the present application discloses methods for administering a modified effector cell to a subject having cancer associated with CD33 overexpression. In some embodiments, this application discloses methods of administering a modified effector cell to a subject having cancer associated with overexpression of CD44, BCMA, CD123, EGFRvIII, folate receptor α, 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, MAGE-A1, h5T4, PSMA, TAG-72 or VEGF-R2. In some cases, the cancer is a metastatic cancer. In other cases, the cancer is recurrent or refractory cancer.

[000389] In some cases, the cancer is a solid tumor or a hematological malignancy. In some cases, cancer is a solid tumor. In other cases, the cancer is a hematological malignancy. In some cases, the cancer is a metastatic cancer. In some cases, the cancer is recurrent or refractory cancer.

[000390] In some cases, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, anal cancer; appendix cancer; bile duct cancer (i.e. cholangiocarcinoma); bladder cancer; a brain tumor; mammary cancer; cervical cancer; colon cancer; cancer with unknown primary origin (CUP); esophageal carcinoma; eye cancer; fallopian tube cancer; gastroenterological cancer; kidney cancer; liver cancer; lungs' cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreas cancer; parathyroid disease; penile cancer; pituitary tumor; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; throat cancer; thyroid cancer; uterine cancer; vaginal cancer; or cancer of the vulva.

[000391] In some cases, the cancer is a hematological malignancy. In some cases, the hematologic malignancy includes lymphoma, leukemia, myeloma, or B-cell malignancy. In some cases, the hematologic malignancy includes lymphoma, leukemia, or myeloma. In some cases, exemplary hematologic malignancies include chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (MLL), high risk CLL, non-CLL/MLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenegrem's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodular marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade B-cell lymphoma, non-Burkitt's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. According to some embodiments of the present invention, the hematologic malignancy includes myeloid leukemia. According to some embodiments of the present invention, the hematologic malignancy includes acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[000392] In some cases, this application discloses methods of administering a modified effector cell described in this application to a subject having a hematological malignancy selected from chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (MLL), high-risk CLL, non-CLL /MLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle zone cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma , nodular marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade non-Burkitt's B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some instances, the present application discloses methods of administering a modified effector cell to a subject having a hematologic malignancy selected from AML or CML.

[000393] In other cases, this application discloses methods of administration to a subject having an infection caused by an infectious disease. An infectious disease may be a disease resulting from a bacterial, viral or fungal infection. In other instances, exemplary viral pathogens include those from the Adenoviridae families, Epstein-Barr virus (EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, BK virus, HSV, HHV virus families, 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, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes and Salmonella.

Doses of modified effector cells

[000394] According to some embodiments of the present invention, a number of modified effector cells are administered to a subject in need thereof, the amount being determined based on the efficacy and potential for inducing cytokine-associated toxicity. In some cases, the number of modified immune effector cells is from about 10 ² to about 10 ⁹ modified immune effector cells/kg. In some cases, the number of modified immune effector cells is from about 10 ³ to about 10 ⁹ modified immune effector cells/kg. In some cases, the number of modified immune effector cells is from about 10 ⁴ to about 10 ⁹ modified immune effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁵ to about 10 ⁹ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁵ to about 10 ⁸ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁵ to about 10 ⁷ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁶ to about 10 ⁹ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁶ to about 10 ⁸ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁷ to about 10 ⁹ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁵ to about 10 ⁶ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁶ to about 10 ⁷ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁷ to about 10 ⁸ modified effector cells/kg. In some cases, the number of modified effector cells is from about 10 ⁸ to about 10 ⁹ modified effector cells/kg. In some cases, the number of modified effector cells is about 10 ⁹ modified effector cells/kg. In some cases, the number of modified effector cells is about 10 ⁸ modified effector cells/kg. In some cases, the number of modified effector cells is about 10 ⁷ modified effector cells/kg. In some cases, the number of modified effector cells is about 10 ⁶ modified effector cells/kg. In some cases, the number of modified effector cells is about 10 ⁵ modified effector cells/kg.

[000395] According to some embodiments of the present invention, the modified effector cells are modified T cells. In some cases, the modified T cells are CAR T cells. In some cases, the number of CAR-T cells is from about 10 ² to about 10 ⁴ CAR-T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁵ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CAR T cells is from about 10 ⁵ to about 10 ⁸ CAR T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁵ to about 10 ⁷ CAR-T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁶ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁶ to about 10 ⁸ CAR-T cells/kg. In some cases, the number of CAR T cells is from about 10 ⁷ to about 10 ⁹ CAR T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁵ to about 10 ⁶ CAR-T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁶ to about 10 ⁷ CAR-T cells/kg. In some cases, the number of CAR-T cells is from about 10 ⁷ to about 10 ⁸ CAR-T cells/kg. In some cases, the number of CAR T cells is from about 10 ⁸ to about 10 ⁹ CAR T cells/kg. In some cases, the number of CAR-T cells is about 10 ⁹ CAR-T cells/kg. In some cases, the number of CAR-T cells is about 10 ⁸ CAR-T cells/kg. In some cases, the number of CAR-T cells is about 10 ⁷ CAR-T cells/kg. In some cases, the number of CAR-T cells is about 10 ⁶ CAR-T cells/kg. In some cases, the number of CAR-T cells is about 10 ⁵ CAR-T cells/kg.

[000396] According to some embodiments of the present invention, CAR T cells are CD19-specific CAR T cells. In some cases, the number of CD19-specific CAR-T cells is from about 10 ² to about 10 ⁹ CAR-T cells/kg. In some embodiments of the present invention, the CAR-T cells are CD19-specific CAR-T cells. In some cases, the number of CD19-specific CAR-T cells is from about 10 ³ to about 10 ⁹ CAR-T cells/kg. In some embodiments of the present invention, the CAR-T cells are CD19-specific CAR-T cells. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁴ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁵ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁵ to about 10 ⁸ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁵ to about 10 ⁷ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁶ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁶ to about 10 ⁸ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁷ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁵ to about 10 ⁶ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁶ to about 10 ⁷ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁷ to about 10 ⁸ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is from about 10 ⁸ to about 10 ⁹ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is approximately 10 ⁹ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is approximately 10 ⁸ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is approximately 10 ⁷ CAR-T cells/kg. In some cases, the number of CD19-specific CAR T cells is about 10 ⁶ CAR T cells/kg. In some cases, the number of CD19-specific CAR-T cells is approximately 10 ⁵ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is approximately 10 ⁴ CAR-T cells/kg. In some cases, the number of CD19-specific CAR-T cells is approximately 10 ³ CAR-T cells/kg. In some cases, the number of CD19-specific CAR T cells is about 10 ² CAR T cells/kg.

[000397] According to some embodiments of the present invention, the modified T cells are modified TCR-T cells. In some cases, the number of modified TCR-T cells is from about 10 ² to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR-T cells is from about 10 ³ to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR-T cells is from about 10 ⁴ to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR-T cells is from about 10 ⁵ to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR-T cells is from about 10 ⁵ to about 10 ⁸ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁵ to about 10 ⁷ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁶ to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁶ to about 10 ⁸ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁷ to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁵ to about 10 ⁶ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁶ to about 10 ⁷ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁷ to about 10 ⁸ TCR cells/kg. In some cases, the number of modified TCR cells is from about 10 ⁸ to about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ⁹ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ⁸ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ⁷ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ⁶ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ⁵ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ⁴ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ³ TCR cells/kg. In some cases, the number of modified TCR cells is about 10 ² TCR cells/kg.

Pharmaceutical compositions and dosage forms

[000398] In some embodiments, this application discloses compositions containing a polynucleotide or polypeptide described herein for administration to a subject. In some cases, modified effector cell compositions encoding a polynucleotide or polypeptide disclosed herein and optionally containing a cytokine and/or an additional therapeutic agent are provided. In some cases, vectors are provided that encode polypeptide constructs for the expression of cell marks for effector cell modification.

[000399] In some instances, pharmaceutical compositions of the modified effector cell or vector encoding the polypeptide constructs and the chimeric antigen receptor are prepared in a conventional manner using one or more physiologically acceptable carriers, including adjuvants and adjuvants that facilitate processing of the active compounds into formulations, which can be used pharmaceutically. The proper composition depends on the chosen route of administration. A brief description of the pharmaceutical compositions described in this application can be found, for example, in Remington: The Science and Practice of Pharmacy, 19th edition. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; ed. Liberman, H.A. and Lachman, L, Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999).

[000400] Pharmaceutical compositions are optionally made in a conventional manner, such as, by way of example only, conventional mixing, dissolving, granulating, drageeing, triturating, emulsifying, encapsulating, encapsulating, or compressing methods.

[000401] According to certain embodiments of the present invention, the compositions may also contain one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric, and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount necessary to maintain the pH of the composition within an acceptable range.

[000402] According to other embodiments of the present invention, the compositions may also contain one or more salts in an amount necessary to bring the osmolality of the composition to an acceptable range. Such salts include salts containing sodium, potassium or ammonium cations and chloride, ascorbate citrate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[000403] The pharmaceutical compositions described herein are administered by any suitable route of administration, including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraarticular, intraperitoneal, or intracranial), intranasal , buccal, sublingual or rectal routes of administration. In some cases, the pharmaceutical composition is formulated for parenteral (eg, intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraarticular, intraperitoneal, or intracranial) administration.

[000404] The pharmaceutical compositions described herein are formulated in any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, paste mixtures, suspensions, and the like, for oral administration by an individual , solid oral dosage forms, aerosols, controlled release formulations, fast melting formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulse formulations release, multiparticulate formulations and mixed immediate release and controlled release formulations. In some embodiments of the present invention, the pharmaceutical compositions are in the form of capsules. According to some variants of implementation of the present invention, the pharmaceutical compositions are made in the form of solutions (for example, for intravenous administration). In some cases, the pharmaceutical composition is formulated as an infusion. In some cases, the pharmaceutical composition is made in the form of an injection.

[000405] The pharmaceutical solid dosage forms described herein optionally contain a compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, a binder, a bulking agent, a suspending agent, a flavoring agent, a sweetening agent, a disintegrating agent, a dispersing agent, a surfactant, a lubricant, a colorant, a diluent, a solubilizer, a wetting agent, a plasticizer, a stabilizer, a penetration enhancer, a wetting agent, an antifoam agent, an antioxidant, a preservative, or one or more combinations thereof.

[000406] In other aspects, the compositions are film-coated using standard coating procedures such as those described in Remington's Pharmaceutical Sciences, 20th ed. (2000). In some embodiments of the present invention, the compositions are formulated into particles (eg, for administration by capsule) and some or all of the particles are coated. In some embodiments of the present invention, the compositions are formulated into particles (eg, for administration by capsule) and some or all of the particles are microencapsulated. In some embodiments of the present invention, the compositions are formulated into particles (eg, for administration by capsule) and some or all of the particles are not microencapsulated or coated.

[000407] According to certain embodiments of the present invention, the compositions of the present invention may also contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[000408] "Proliferative disease", as referred to in this application, means a complex concept, according to which excessive cell proliferation and renewal of the cell matrix contribute significantly to the pathogenesis of a number of diseases, including cancer.

[000409] As used herein, the term "patient" refers to a mammalian subject who has been diagnosed with and suspected of having or developing a physiological condition, such as cancer, an autoimmune condition, or infection. In some embodiments of the present invention, the term "patient" refers to a mammalian subject with a higher than average likelihood of developing cancer. Patient examples may include humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents, and other mammals that may benefit from the therapies described herein. Examples of human patients may include male and/or female.

[000410] "Introduction" is referred to in this application as providing the compositions according to the present invention to the patient. For example, but not limited to, administration of the composition, e.g., by injection, may be by intravenous (IV) injection, subcutaneous (SC) injection, intradermal (IV) injection, intraperitoneal (IP) injection, or intramuscular (IM) injection. One or more of these paths may be used. Parenteral administration can be carried out, for example, by bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route. In addition, administration can also be accomplished by surgical placement of a bolus or cell mass, or placement of a medical device.

[000411] A "patient in need thereof" or "subject in need thereof" is referred to herein as a patient or subject who is diagnosed or suspected of having a disease or disorder, such as, but not limited to, a proliferative disorder such like cancer. According to one embodiment of the present invention, the patient or subject has or is likely to develop a solid tumor or leukemia. According to some variants of implementation of the present invention, leukemia can be, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML).

[000412] The compositions of the present invention may contain host cells expressing the nucleic acid sequences of the present invention, or a vector containing the nucleic acid sequence of the present invention, in an amount effective to treat or prevent proliferative disorders. In this application, the terms "treatment", "treating" and the like refer to obtaining the target pharmacological and/or physiological effect. In embodiments, the effect is therapeutic, i. the effect partially or completely cures the disease and/or an undesirable symptom associated with the disease. To this end, the method of the present invention comprises administering a "therapeutically effective amount" of a composition comprising host cells expressing a nucleic acid sequence of the present invention or a vector containing nucleic acid sequences of the present invention.

[000413] "A therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on such factors as the pathological condition, age, sex and weight of the individual and the ability of the nucleic acid sequences of the present invention to elicit a targeted response in the individual.

[000414] In another embodiment, the pharmacological and/or physiological effect may be "prophylactic", ie. the effect completely or partially prevents the disease or its symptom.

[000415] A "prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result (eg, preventing the onset of a disease).

[000416] "Antifoam agents" reduce foaming during processing, which can lead to coagulation of aqueous dispersions, the formation of bubbles in the finished film, or generally interfere with processing. Exemplary antifoam agents include silica emulsions or sorbitan sesquioleate.

[000417] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulphite, and tocopherol. According to certain variants of implementation of the present invention, antioxidants increase chemical stability, if necessary.

[000418] The use of antioxidants, metal chelating agents, thiol-containing compounds, and other general stabilizing agents may be beneficial to the formulations described herein. Examples of such stabilizing agents include, but are not limited to: (a) from about 0.5% to about 2% wt./about. glycerol, (b) from about 0.1% to about 1% wt./about. methionine, (c) from about 0.1% to about 2% wt./about. monothioglycerol, (d) from about 1 mm to about 10 mm EDTA, (e) from about 0.01% to about 2% wt./about. ascorbic acid, (f) from 0.003% to about 0.02% wt./about. polysorbate 80, (g) from 0.001% to about 0.05% wt./about. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[000419] "Binders" impart cohesive properties and include, for example, alginic acid and its salts; cellulose derivatives such as carboxymethylcellulose, methylcellulose (eg Methocel®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (eg Klucel®), ethylcellulose (eg Ethocel®) and microcrystalline cellulose (eg Avicel®); microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonites; gelatin; polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, sugar such as sucrose (eg Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (eg Xylitab®) and lactose; natural or synthetic gum such as gum arabic, tragacanth, gum gattia, psyllium husk mucus extract of the genus Plantago, polyvinylpyrrolidone (e.g. Polyvidone® CL, Kollidon® CL, Polyplasdone® XL-10), larch arabogolactan, Veegum®, polyethylene glycol, waxes, sodium alginate and the like.

[000420] "Carrier" or "carrier materials" include any commonly used excipients in the pharmaceutical field and should be selected based on compatibility with the compounds disclosed in this application, such as compounds of ibrutinib, and an anticancer agent, and properties of the release profile of the target drug forms. Exemplary transport materials include, for example, binders, suspending agents, disintegrants, bulking agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "Pharmaceutical compatible carrier materials" may include, but are not limited to, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, lecithin soy, taurocholic acid, phosphatidylcholine, sodium chloride, tribasic calcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, for example, Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; ed. Liberman, H.A. and Lachman, L, Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999).

[000421] "Dispersing agents" and/or "viscosity modulating agents" include materials that allow the diffusion and homogeneity of a drug to be controlled using liquid media or a granulation process or a mixing process. According to some variants of implementation of the present invention, these agents also improve the efficiency of coating or destruction of the matrix. Exemplary diffusion/dispersing agents include, for example, hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and carbohydrate-based dispersants such as, for example, hydroxypropyl celluloses (for example, HPC, HPC-SL and HPC-L), hydroxypropyl methylcelluloses (e.g. HPMC K100, HPMC K4M, HPMC K15M and HPMC K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate-stearate (AHPMC), non-crystalline cellulose , magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone/vinyl acetate copolymer (S630), polymer of 4-(1,1,3,3-tetramethylbutyl)phenol with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68®, F88® and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, also known as poloxamine 908®, which is a tetrafunctional block copolymer obtained by sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsipany, NJ, USA)), polyvinylpyrrolidone K12 , polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate (S-630) copolymer, polyethylene glycol, for example, polyethylene glycol may have a molecular weight of from about 300 to about 6000, or from about 3350 to about 4000, or from about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums such as, for example, tragacanth and gum arabic, guar gum, xanthans, including xanthan gum, sugars, celluloses, such as, for example, sodium carboxymethylcellulose, methylcellulose, carboxymethylcellulose sodium, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans, and combinations thereof. Plasticizers such as cellulose or triethylcellulose can also be used as dispersing agents. Dispersing agents particularly suitable for liposomal dispersions and spontaneously emulsifying dispersions include dimyristoylphosphatidylcholine, natural egg phosphatidylcholine, natural egg phosphatidylglycerin, cholesterol, and isopropyl myristate.

[000422] Combinations of one or more erosion-facilitating agents with one or more diffusion-facilitating agents can also be used in the compositions of the present invention.

[000423] The term "diluent" refers to chemicals that are used to dilute the compound of interest prior to delivery. Diluents can also be used to stabilize compounds as they can provide a more stable environment. Salts dissolved in buffered solutions (which may also provide pH control or maintenance) are used in the art as diluents, including, but not limited to, phosphate buffered saline. In certain embodiments of the present invention, diluents increase the overall weight of the composition to facilitate compression, or provide sufficient overall weight for a homogeneous mixture to fill capsules. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®, dibasic calcium phosphate, dibasic calcium phosphate dihydrate; trisubstituted calcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed dry matter of cereals, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite and the like.

[000424] "Filling agents" include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol and the like.

[000425] "Lubricants" and "gliders" are compounds that prevent, reduce, or inhibit adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, a hydrocarbon such as mineral oil, or a hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal salts and alkaline earth metals such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerin, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g. PEG-4000 ) or methoxy polyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silicon dioxide such as Syloid™, Cab-O-Sil®, starch such as cornstarch, silicone oil, surfactant and the like.

[000426] "Plasticizers" are compounds used to soften the material for microencapsulation or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG300, PEG400, PEG600, PEG1450, PEG3350 and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. According to some variants of implementation of the present invention, plasticizers can also act as dispersing agents or wetting agents.

[000427] "Solubilizers" include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol and dimethyl isosorbide and the like.

[000428] "Stabilizers" include compounds such as any antioxidant agents, buffers, acids, preservatives, and the like.

[000429] "Suspension agents" include compounds such as polyvinylpyrrolidone, for example, polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, for example, polyethylene glycol may have a molecular weight of from about 300 to about 6000 , or from about 3350 to about 4000, or from about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums such as, for example, gum tragacanth and gum arabic, guar gum, xanthans, including xanthan gum, sugars, cellulose derivatives such as, for example, sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like .

[000430] "Surfactants" include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, for example, Pluronic® (BASF) and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, for example, octoxynol 10, octoxynol 40. According to some embodiments of the present invention, surfactants may be included to increase physical stability or for other purposes.

[000431] "Viscosifying agents" include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate-stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, gum arabic, chitosans, and combinations thereof.

[000432] "Wetting agents" include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, in vitamin E TPGS, ammonium salts and the like.

Kits / product of industrial production

[000433] In certain embodiments, this application discloses commercial kits and articles for use with one or more of the methods described herein. Such kits contain a carrier, package or container that is divided into compartments to accommodate one or more containers such as vials, test tubes, and the like, each container containing one or more separate elements for use in the method described in this application. Suitable containers include, for example, bottles, vials, syringes and test tubes. In one embodiment, the containers are made from various materials such as glass or plastic.

[000434] The industrial articles of the present invention comprise packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, vials, pouches, containers, bottles, and any packaging material suitable for the selected formulation and intended route of administration and treatment.

[000435] For example, the container(s) contains(at) cells encoding polypeptide constructs expressing one or more truncated non-immunogenic polypeptides described in this application (for example, CD20 or CD52). Optionally, the cells may further contain one or more heterologous genes, for example, a gene encoding a CAR, a T cell receptor, and/or a cytokine. Such kits do not necessarily contain an identifying description or label, or instructions relating to their use in the methods described in this application.

[000436] The kit typically contains labels listing contents and/or instructions for use, as well as package inserts with instructions for use. Typically, a set of instructions is also included.

[000437] According to some embodiments of the present invention, the label is on or associated with the container. According to one embodiment of the present invention, a label is on a container if the letters, numbers, or other symbols that make up the label are affixed, molded, or etched onto the container itself; a label is associated with a container if it is present inside a container or holder that also holds the container, for example as a package insert with instructions for use. According to one embodiment of the present invention, the label is used to indicate that the content is to be used for a particular therapeutic application. The label also contains instructions for using the contents, for example, in the methods described in this application.

SEQUENCES

[000438] The following is a typical list of certain sequences included in the implementation options presented in this application.

EXAMPLES

[000439] The examples are provided for illustrative purposes only, and not to limit the scope of the claims presented in this application.

Example 1 Expression and activity of a truncated CD20 (CD20t) polypeptide construct as a cell tag

[000440] HEK-293T cells were transfected with wild-type CD20, truncated CD20 cell labels, and a positive control for the anti-CD20 antibody rituximab. Expression was measured by flow cytometry using rituximab. As shown in FIG. 1, a truncated CD20 variant, CD20t1 (SEQ ID NO: 109), showed stable expression when detected with rituximab relative to the CD20t4 variant (SEQ ID NO: 115). These data indicate that only specific truncations in the endogenous CD20 polypeptide may be compatible with the use of CD20t as a cell label.

[000441] Human PBMCs were nucleofected using the Sleeping Beauty transposon encoding CAR and the CD20t1 cell tag, as well as Sleeping Beauty transposase plasmids. Co-expression of CAR and CD20t was measured by flow cytometry. FIG. 2 shows that CD20t1 (SEQ ID NO: 109; y-axis) and CAR (x-axis) are co-expressed from the CAR-CD20t1 construct compared to untransfected cells.

[000442] Anti-CD20 specific antibody-induced ADCC of truncated CD20 cell labels

[000443] The Jurkat reporter cell line was modified to stably express the CD20t1 cell marker. CD20t1 expression was confirmed by flow cytometry using rituximab staining. The ability of rituximab to specifically eliminate CD20t1-expressing Jurkat target cells was measured in an in vitro cytotoxicity assay. FIG. 3 shows rituximab-induced ADCC activity of a cell line expressing CD20t1. In comparison, treatment with cetuximab directed against EGFR had no effect on ADCC activity.

Example 2 Expression of a Truncated HER1 (HER1t) Polypeptide Construct as a Cell Label

Expression of chimeric cell marks

[000444] New chimeric cell labels were generated using domain III of the HER1 gene and domain IV of the HER2, 3 or 4 genes. HER1-ErBB4 chimeric cell labels were expressed in primary human cells. Domain III of the HER1 gene was genetically hybridized with domain IV of the JM-a or JM-b variant of the ErbB4 gene and the TM domain of the ErbB4 gene to obtain chimeric cell marks. The signal peptide GM-CSF-alpha was used as a signal peptide to target chimeric cell labels to the cell surface. HER1-ErbB4 cell tags (truncated EGFR-ErbB4 (JM-a) corresponding to SEQ ID NO: 101 and truncated EGFR-ErbB4 (JM-b) corresponding to SEQ ID NO: 105) were cloned into the pCDNA3.1 vector backbone together with a control HER1t cell label and transfected by electroporation into primary human PBMCs. Expression of HER1-ErbB4 cell marks was confirmed in CD3 ⁺ T cells by flow cytometry using a specific anti-HER1 antibody, cetuximab. As shown in Figure 4D, transduced T cells expressed high levels of chimeric cell marks after transfection. The results of staining with isotype antibody, as well as non-transfected control cells, indicated the specificity of staining of cell labels with cetuximab.

Expression of truncated HER1t cell marks

[000445] Various truncated cell labels were designed as depicted in the scheme shown in FIG. 4A. Truncated HER1t variants (HER1t1 corresponding to SEQ ID NO: 57, HER1t2 corresponding to SEQ ID NO: 59, HER1t3 corresponding to SEQ ID NO: 61, HER1t4 corresponding to SEQ ID NO: 63, HER1t5 corresponding to SEQ ID NO: 65, HER1t6 corresponding to SEQ ID NO: 67, and HER1t7 corresponding to SEQ ID NO: 69), together with a control HER1t cell tag, were cloned into the pCDNA3.1 vector backbone for expression testing. These expression vectors were transfected by electroporation into primary human PBMCs. Expression of truncated cell labels was confirmed in CD3 ⁺ T cells by flow cytometry using the specific anti-HER1 antibody, cetuximab. As shown in Figure 4E, T cells expressed high levels of cell marks after transfection. The results of staining with isotype antibody, as well as non-transfected control cells, indicated the specificity of staining of cell labels with cetuximab.

[000446] Self-inactivating lentiviral vectors encoding either CAR alone or CAR and a truncated HER1t1 cell tag were generated to assess co-expression of both genes. A total population of activated human T cells were transduced with lentiviral vectors, and expression of both CAR and HER1t1 was measured post-transduction by flow cytometry using a CAR-specific antigen-Fc fusion protein and an anti-HER1 antibody, cetuximab, respectively. As shown in the right panel of FIG. 5A and FIG. 5B, transduced T cells coexpressed both CAR (x-axis) and HER1t (y-axis). In the left panel of FIG. 5A shows the minimal non-specific staining seen in non-transduced T cells. In the left panel of FIG. 5B shows expression of CAR, but not HER1t, when T cells are transduced with a lentiviral vector encoding only CAR.

Analysis of cell lines expressing a truncated HER1t cell label by Western blotting

[000447] The SUP-T1 cell line was genetically modified to express HER1t1 (SUP-T1/HER1t1). The SUP-T1/HER1t1 cell line was FACS sorted for either high (high) or low (low) HER1t1 expression as measured by flow cytometry. Expression of the truncated HER1t cell marker was confirmed in the SUP-T1/HER1t1 (high) and SUP-T1/HER1t1 (low) cell lines by Western blotting. The anti-HER1 antibody, cetuximab, was incubated with extracts of the SUP-T1/CAR/HER1t1 cell line or control cell line (Jurkat and A431) to allow the antibody to bind to HER1-derived cell marker proteins. The antibody/antigen complex was then precipitated using protein A/G bound agarose beads. This sample was then separated by SDS-PAGE for analysis by Western blot using anti-HER1 antibody. Western blot results shown in FIG. 8 confirmed HER1t1 expression in SUPT1/HER1t1 cell lines. HER1t1 intensity is lower in the SUP-T1/HER1t1 cell line (low) compared to the SUP-T1/HER1t1 cell line (high). Full length HER1 was detected in the A431 positive control cell line. In the unmodified Jurkat cell line, HER1 expression was not detected. Fig. 8: Lane 1: i.p. antibody only; Lane 2: Jurkat cages only; Lane 3: SUPT1/HER1t1 (high HER1t1); Lane 4: SUPT1/HER1t1 (lower levels of HER1t1); Lane 5: A431 cells expressing full length HER1; Lane 6: marker. The bold arrow indicates protein precipitated by cetuximab in lanes except lane 5 where it refers to full-length HER1.

Example 3 Anti-HER1 antibody-induced ADCC and CDC of cells expressing a truncated HER1t cell label

[000448] The functionality of the HER1t1 cell label in the ADCC assay was assessed using a MK cell line. The original NK cell line was modified to express the CD16 receptor variant to induce ADCC. The CD16 ⁺ NK cell line was further modified to co-express CAR and the HER1t1 cell label. The original NK, CD16 ⁺ NK and CD16 ⁺ /CAR ⁺ /HER1t1 ⁺ NK cell lines were analyzed for ADCC in the presence of either an anti-HER1 antibody, cetuximab, or alemtuzumab as a positive control capable of binding CD52 on NK cells. As shown in FIG. 6, ADCC was observed only when NK cells expressed CD16, as expected, since antibody binding to CD16 induced effector function. In addition, alemtuzumab was able to induce ADCC in NK cells, regardless of their expression of the HER1t cell marker, provided they expressed CD16. In contrast, cetuximab induced ADCC only when both HER1t1 and CD16 were expressed, suggesting specificity.

[000449] The SUP-T1 reporter cell line (human T lymphocyte line) was genetically modified to co-express CAR and HER1t1 (SUP-T1/CAR/HER1t1). The SUP-T1/CAR/HER1t1 cell line was FACS sorted for high (high) or medium (medium) HER1t1 expression as measured by flow cytometry to assess the effect of cell surface label density on ADCC using an anti-HER1 antibody. FIG. 7A shows CAR and HER1t1 expression levels in sorted SUP-T1/CAR/HER1t1 populations. In the left panel of FIG. 7A shows isotype antibody staining only, middle panel shows high HER1t expression, and right panel shows average HER1t expression by flow cytometry. Since the CAR and HER1t1 genes are expressed from the same transcript, HER1t1 cell sorting also affected CAR expression in a similar way. SUP-T1/CAR/HER1t1 cell lines with high and medium expression levels were tested in the ADCC assay using cetuximab or non-specific rituximab as controls. For the analysis used the ratio of effector cells (E) and HER1t1 ⁺ target cells (T), constituting 5:1. ADCC was quantified as the relative induction of reporter gene expression. As shown in FIG. 7B, cetuximab specifically induced ADCC in a cell line expressing HER1t1 in a dose dependent manner. In addition, ADCC induction depended on the level of HER1t1 expression on the cell surface.

[000450] Donor human PBMCs were transfected by electroporation using two Sleeping Beauty transposon vectors to express CD19 CAR and HER1t1 cell tag along with SB11 transposase to redirect T cell specificity. The day after transfection (Day 1), cells were counted and CAR expression was measured by flow cytometry. CAR-T cells were stimulated with AaPC either γ-irradiated (100 Gy) or treated with mitomycin C in a 1:1 ratio for 4 cycles of stimulation. The AaPC cells used were K562-AaPC expressing the CD19 antigen. Cultures were supplemented with IL-21 (30 ng/ml) for the first round of stimulation only, followed by recombinant human IL-2 (50 IU/ml) and IL-21 (30 ng/ml) (Pepro Tech) for the remaining stimulations. T cell cultures were phenotyped at the end of each stimulation cycle, which typically lasted 7 days. Cultures were phenotyped to determine CAR expression by multivariable flow cytometry using protein L or an anti-idiotypic antibody that recognizes CD19 CAR. The ability of cetuximab to specifically eliminate CD19 GAR ⁺ /HER1t1 ⁺ T cells under in vitro conditions was tested in the ADCC and complement-dependent cytotoxicity (CDC) assays. CSFE-labeled CD19 CAR ⁺ /HER1t1 ⁺ target T cells (T) (5×10 ⁴ cells/well) were incubated with a CD16 ⁺ NK effector cell line (E) (2.5×10 ⁵ cells/well) at an E:T ratio of 5:1 with 5 µg/ml cetuximab or rituximab for 2-24 hours in triplicate. For CSC analysis, 10% rabbit serum, either heat-inactivated (HI) or non-inactivated, and 30% human serum, either heat-inactivated (HI) or non-inactivated, were used. Cells were stained with DAPI and data was obtained using the iQUE Screener plus instrument. The number of living cells was recorded for each condition. As shown in FIG. 9 (left panel), cetuximab induced specific cytotoxicity of CD19 CAR ⁺ /HER1t1 ⁺ T cells, as evidenced by the low number of viable cells in the ADCC assay. As shown in FIG. 9 (right panel), cetuximab induced specific cytotoxicity of CD19 CAR ⁺ /HER1t1 ⁺ T cells, as evidenced by the low number of viable cells in the CSC assay, in which human or rabbit sera were not heat-inactivated.

Example 4 Next Generation Switch-to-Kill/Cell Label Constructs Enhance ADCC and CDC

[000451] FIG. 10 is a schematic diagram comparing the design of first generation and next generation polypeptide constructs containing truncated cell tags. Truncated first generation cell labels (top) contain a truncated variant and a non-dimerizable transmembrane domain (TM) connected by an optional peptide linker. Truncated next generation cell labels (bottom panel) contain a multimerization domain to produce cell labels that are capable of multimerizing on the cell surface. In the example shown in the bottom panel of FIG. 10, a transmembrane domain (TM-A) capable of homodimerization is used to induce the formation of cell label dimers on the cell surface. While homodimerization of cell surface polypeptides has been shown, it is also possible for cell surface heterodimers to form if cells are modified to co-express polypeptide constructs having different cell surface polypeptides. In both first generation and next generation polypeptide constructs, the truncated variant antibody or other binding domain recognizes and binds to the truncated variant. For such next generation constructs, dimerization of cell surface polypeptides can increase avidity and enhance the signaling effect induced by antibody binding compared to first generation constructs, as well as improve cell purification and sorting using a multimeric cell label.

[000452] Donor human PBMCs were transfected (Day 0) by electroporation using Sleeping Beauty transposon vectors designed to co-express CD19 CAR and first or next generation HER1t cell marks along with SB11 transposase to redirect T cell specificity. The day after transfection (Day 1), cells were counted and CAR expression was measured by flow cytometry. CAR-T cells were stimulated with AaPC, either γ-irradiated (100 Gy) or treated with mitomycin C, in a 1:1 ratio for 4 cycles of stimulation. The AaPC cells used were K562-AaPC expressing the CD19 antigen. Cultures were supplemented with IL-21 (30 ng/ml) only for the first round of stimulation, followed by recombinant human IL-2 (50 IU/ml) and IL-21 (30 ng/ml) (Pepro Tech) for the remaining stimulations. T cell cultures were phenotyped at the end of each stimulation cycle, which typically lasted 7 days. Cultures were phenotyped for CAR expression by multivariable flow cytometry using either the L protein or an anti-idiotypic antibody that recognized CD19 CAR and an anti-HER1 antibody, cetuximab, that recognized various truncated HER1t cell marks. FIG. 11 shows that CD19 CAR expression does not change over time in next-generation polypeptide constructs containing HER1t variants (e.g., HER1t8 corresponding to SEQ ID NO: 71, HER1t9 corresponding to SEQ ID NO: 75, and HER1t10 corresponding to SEQ ID NO: 79) compared to a first generation polypeptide construct (HER1t1 corresponding to SEQ ID NO: 57) and a control cell line that does not express HER1t. The percentage of CD3 ⁺ T cells expressing the CD19 CAR in different cultures is shown in Table 4.

[000453] FIG. 12 shows that cell lines expressing next generation polypeptide constructs containing HER1t variants with a transmembrane dimerization domain (HER1t8 variants corresponding to SEQ ID NO: 71, HER1t9 corresponding to SEQ ID NO: 75, and HER1t10 corresponding to SEQ ID NO: 79) , have similar levels of HER1t expression over time as the line expressing the first generation HER1t polypeptide construct (HER1t1 corresponding to SEQ ID NO: 57). In a culture transfected with only the CAR transposon, HER1t expression was not detected. Table 5 shows the percentage of CD3 ⁺ T cells expressing HER1t variants in different cultures, while Table 6 shows the mean fluorescence intensity (MFI) of HER1t at days 14, 21 and 28 from FIG. 12.

[000454] CD19 CAR-T cells expressing next generation HER1t variants were tested for cetuximab-induced ADCC in an in vitro assay and compared for potency with a first generation HER1t construct. CD19 CAR-T target (T) cells were labeled with CFSE at a concentration of 0.0005 μM for CAR-T cells expressing HER1t variants and 0.01 μM for CAR-T cells without HER1t. Target cells were suspended at 0.8×10 ⁶ cells/ml, mixed at a ratio of 1:1 and plated at 4×10 ⁴ cells/50 μl/well (in each case 2×10 ⁴ cells/50 μl/ hole). The effector CD16 ⁺ NK cells (E) were then harvested, washed and resuspended at 0.2×10 ⁶ cells/ml. The cells were then seeded at 2×10 ⁴ cells/100 μl/well (E:T=0.5:1). Rituximab or cetuximab antibodies were prepared at a concentration of 0.4 µg/mL along with a control solution that lacked antibody, and the solutions were added to the wells as follows: (i) Rituximab control antibody (anti-CD20 antibody): 50 µL/well at final concentration 0.1 µg/ml in each well; (ii) cetuximab (anti-HER1 antibody): 50 µl/well at a final concentration of 0.1 µg/ml in each well; (iii) control solution (no antibodies): 50 µl/well at a final concentration of 0 µg/ml in each well. The final volume in each well was adjusted to 200 μl. The solutions were well mixed and cultured for 4, 8, 16 and 24 hours. After incubation, cells were washed and stained with CD3-BW86 in the presence of a human Fc block. The cells were then washed twice with FACS buffer, resuspended in 50 μl of FAC buffer containing 7AAD for 10 minutes, and then analyzed. FIG. 13A-B show that next-generation HER1t polypeptide constructs containing potentially dimerized HER1t variants on cell surface polypeptides (HER1t8, HER1t9, and HER1t10) mediate improved ADCC compared to polypeptide constructs containing a non-dimerizable transmembrane domain (HER1t control) in the presence of cetuximab. .

[000455] In vitro studies were performed to confirm the ability of cetuximab to induce ADCC against CD19CAR-mbIL15-T cells. CD19CAR-mbIL15-T cells were generated by electrotransfer of CD19CAR and mbIL15-HER1t transposons and SB11 transposase. Genetically modified T cells were subjected to quantitative propagation in ex vivo conditions on irradiated CD19 ⁺ feeder cells for analysis of ADCC. Due to the bicistronic design of the mbIL15-HER1t transposon, CD19CAR-mblL-15-T cells co-expressed HER1t1 while expressing mbIL15. CD19 CAR ⁺ mbIL15-HER1tneg Negative control T cells lacking mbIL15-HER1t expression, generated by transfection of CD19 CAR transposon and SB11 transposase, were quantitated ex vivo using the same CD19 ⁺ feeder cells. Expanded allogeneic NK cells that express endogenous FcR were used as effector cells and co-cultured overnight with CAR ⁺ labeled T cells at a 10:1 E:T ratio in the presence of 10 µg/mL cetuximab or an anti-CD20 antibody ( rituximab), which served as a negative control. mbIL15-HER1t ⁺ T cells remaining in culture after antibody treatment were determined by flow cytometry to calculate percent kill. The addition of cetuximab resulted in the elimination of >90% of the mbIL15-HER1t ⁺ CD19-mbIL15-CAR T cell population (Figure 14). Rituximab showed a lower rate of non-specific elimination of CD19-mbIL15-CAR-T cells. Cetuximab did not show a significant level of lysis of CAR ⁺ mbIL15-HER1tneg T cells, which confirms a HERH-specific mechanism of action. The 10 µg/mL cetuximab concentration used in this experiment is within the range previously reported in patients treated with cetuximab. These data support the use of cetuximab to deplete CD19CAR ^- mbIL15-T cells, if necessary due to the development of adverse clinical effects.

[000456] An in vivo study was performed on NSG mice. On day 0, 5E6 CAR-HER1t1 T cells and 5E6 KHYG-1-CD16high cells were injected intraperitoneally (ip) into each mouse. On the same day, mice were randomized and injected with saline or cetuximab (0.5 mg: i.p.). Peritoneal lavage was collected on day 1 and evaluated by flow cytometry to estimate the frequency of CAR-HER1t1 T cells in mice in both groups. The absolute number of CAR-HER1t1 T cells was also counted. Data (not shown) demonstrate the ability of cetuximab to specifically eliminate CAR-T cells expressing the HER1t1 tag, in contrast to saline treated mice.

[000457] While preferred embodiments of the present invention have been presented and described herein, those skilled in the art will appreciate that such embodiments are provided by way of example only. Numerous variations, modifications and substitutions will now become apparent to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments described herein, or combinations of one or more of these embodiments or the aspects described therein, may be used in the practice of the present invention. The following claims are intended to define the scope of the present invention, thereby including methods and structures within the scope of these claims and their equivalents.

Claims (41)

1. A polypeptide construct for providing a modified cell with a cell label, said polypeptide construct contains a cell surface polypeptide that is capable of binding to a binding partner and leading to depletion of said modified cell, said cell surface polypeptide contains: a) a truncated non-immunogenic HER1 polypeptide containing: HER1 domain III, which contains an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 200; and a truncated HER1 domain IV that contains an amino acid sequence that is at least 85% identical to the sequence according to any of SEQ ID NOs: 203-209; b) a truncated non-immunogenic CD20 polypeptide that contains an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 109; c) a truncated non-immunogenic CD52 polypeptide that contains an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 143; and/or d) a truncated non-immunogenic LNGFR polypeptide that contains an amino acid sequence that is at least 85% identical to the amino acid sequence according to any of SEQ ID NOs: 156, 158 and 160. 2. The polypeptide construct according to claim 1, characterized in that said cell surface polypeptide contains a truncated non-immunogenic HER1 polypeptide containing: HER1 domain III, which contains an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 200; and a truncated HER1 domain IV that contains an amino acid sequence that is at least 85% identical to the sequence according to any of SEQ ID NOs: 203-209. 3. The polypeptide construct according to claim 2, characterized in that said domain III of HER1 contains an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 200; and said truncated HER1 domain IV contains an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 203-209. 4. Polypeptide design according to any one of paragraphs. 1-3, containing an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 57. 5. A polypeptide construct according to claim 1, characterized in that said cell surface polypeptide contains a truncated non-immunogenic CD20 polypeptide that contains an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 109. 6. A polypeptide construct according to claim 1, characterized in that said cell surface polypeptide contains a truncated non-immunogenic CD52 polypeptide that contains an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 143. 7. Polypeptide design according to any one of paragraphs. 1 or 2, additionally containing a transmembrane domain. 8. A polypeptide construct according to claim 7, wherein said transmembrane domain is a non-HER1 transmembrane domain. 9. A polypeptide construct according to claim 7 or 8, further comprising a peptide linker for connecting said cell surface polypeptide and said transmembrane domain. 10. Polypeptide design according to any one of paragraphs. 7-9, characterized in that said transmembrane domain is capable of inducing dimerization of said cell surface polypeptide. 11. The polypeptide construct of claim 10, wherein said cell surface polypeptide comprises a truncated non-immunogenic HER1 polypeptide that is at least 85% identical to a sequence according to any one of SEQ ID NOs: 211-217. 12. A polypeptide construct according to claim 10, characterized in that said cell surface polypeptide further comprises a truncated non-immunogenic CD20 polypeptide containing an amino acid sequence that is at least 85% identical to the sequence according to any of SEQ ID NOs: 218 - 220. 13. A polypeptide construct according to claim 1, characterized in that said cell surface polypeptide comprises a truncated non-immunogenic LNGFR polypeptide containing an amino acid sequence that is at least 95% identical to the sequence according to any of SEQ ID NO: 156, SEQ ID NO: 158 and SEQ ID NO: 160. 14. Polypeptide design according to any one of paragraphs. 7-12, characterized in that said transmembrane domain can form either a homodimer or a heterodimer with a complementary dimerization domain. 15. Polypeptide design according to any one of paragraphs. 7-12 and 14, characterized in that said transmembrane domain contains at least one cysteine residue. 16. Polypeptide design according to any one of paragraphs. 7-12, 14 and 15, characterized in that said transmembrane domain contains: a glycophorin A transmembrane domain or a functional variant thereof; chimeric transmembrane domain of glycophorin A-integrin β3 or a functional variant thereof; or the transmembrane domain of CD3-zeta. 17. Polypeptide construct according to any one of paragraphs. 1-4, 7-9 or 11 containing: a) a truncated non-immunogenic HER1 polypeptide capable of binding an anti-HER1 antibody and consisting essentially of HER1 domain III and a truncated HER1 domain IV; b) CD28 transmembrane domain; And c) a peptide linker for linking said truncated non-immunogenic HER1 polypeptide to said CD28 transmembrane domain. 18. Polynucleotide encoding a polypeptide construct according to any one of paragraphs. 1-17. 19. An expression vector containing a polynucleotide according to claim 18. 20. A modified immune effector cell for use in adoptive cell therapy, wherein said modified immune effector cell comprises the polynucleotide of claim 18 and a chimeric antigen receptor or T cell receptor. 21. Modified immune effector cell according to claim 20, additionally containing membrane-bound IL-15, containing: a) IL-15 or its functional variant; And b) IL-15α or its functional variant. 22. A modified immune effector cell according to claim 20 containing a chimeric antigen receptor. 23. A modified immune effector cell according to claim 21 containing a chimeric antigen receptor. 24. The modified immune effector cell according to claim 23, characterized in that said chimeric antigen receptor 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-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII , CD123 and/or VEGF-R2. 25. A modified immune effector cell according to claim 20, characterized in that said cell is a T cell or a natural killer (NK) cell. 26. A method for depleting a modified cell expressing a polypeptide construct according to claim 1 in a subject that has been provided with said modified cell, said method comprising: providing said subject with a binding partner that binds to said polypeptide construct, thereby depleting said modified cell. 27. The method of claim 26, wherein said binding partner is rituximab, cetuximab, futuximab, depatuxizumab, imgatuzumab, laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab, zalutumumab, tositumomab, veltuzumab, afutuzumab, blontuvetmab, or both. inutuzumab. 28. A method of treating cancer, comprising administering a modified immune effector cell according to claim 20 to a subject in need thereof, wherein said cancer is associated with overexpression 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, MAGE-A1, h5T4, PSMA , TAG-72, EGFRvIII, CD123 and/or VEGF-R2. 29. The method of claim 28 wherein said modified immune effector cell is administered to said subject in an amount of from about ¹⁰⁴ to about ¹⁰⁹ modified effector cells/kg. 30. The method of claim 29 wherein said modified immune effector cell is administered to said subject in an amount of from about ¹⁰⁴ to about ¹⁰⁷ modified effector cells/kg. 31. The method of claim 28, wherein said cancer is associated with overexpression of CD19, CD33, ROR1 and/or MUC-16. 32. The method of claim 28, wherein said cancer is a hematological malignancy, breast cancer, fallopian tube cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, or prostate cancer.

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