Engineered T cell receptors and engineered immune cells expressing engineered T cell receptors

Abstract

In some embodiments, engineered T cell receptors (TCRs) are provided that comprise an engineered CD3zeta subunit and / or a target-recognizing TCR subunit. The engineered CD3zeta subunit comprises a co-stimulatory region operably linked to or incorporated into an intracellular domain of a mammalian CD3zeta component. The engineered target-recognizing TCR subunit comprises an antigen-binding region or ligand as a target-recognizing portion operably linked to or incorporated into an extracellular domain of any of the mammalian TCR [alpha], TCR [beta], CD3 [epsilon], CD3 [gamma], or CD3 [delta] subunits, or a functional portion or variant thereof. Some embodiments of the engineered target recognition TCR subunit comprise a truncated CD3 epsilon subunit lacking its intracellular domain, and optionally further comprise at least one functional region. The present disclosure also provides immune cells expressing any engineered TCR subunits, where some immune cells exhibit improved properties, including more specific and more potent cytotoxicity against target cells, and advantageously safer therapeutic profiles.

Description

[0001] Priority requirements

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 477,071, filed December 23, 2022, and U.S. Provisional Application No. 63 / 613,545, filed December 21, 2023. The entire contents of the foregoing applications are incorporated herein by reference. Technical Field

[0003] This disclosure relates to engineered T-cell receptors (TCRs) and engineered immune cells expressing said engineered T-cell receptors. In some embodiments, the engineered T-cell receptor comprises an engineered CD3 zeta subunit and / or an engineered CD3epsilon subunit. In some embodiments, the engineered T-cell receptor comprises an engineered target-recognizing T-cell receptor (TCR) subunit, wherein said target-recognizing moiety is a ligand moiety. Background Technology

[0004] T-cell receptors (TCRs) are protein complexes expressed on the surface of T lymphocytes that are typically responsible for recognizing fragments of antigens that bind to the major histocompatibility complex (MHC).

[0005] T cell receptors typically consist of membrane-anchored heterodimers (i.e., TCR alpha (TCRα or TCRRa) and beta (TCRβ or TCRb) chains), each composed of a variable (V) region, a constant (C) region, a transmembrane region, and a short cytoplasmic tail. The variable region contains a hypervariable sequence and is responsible for specific binding to the antigen / MHC complex. However, the TCR heterodimer itself cannot transduce activation signals; it depends on the CD3 signaling transduction complex that interacts with it. The CD3 complex consists of one CD3 gamma (CD3γ or CD3g) chain, one CD3 delta (CD3δ or CD3d) chain, two CD3 epsilon (CD3ε or CD3e) chains, and two CD3 zeta (CD3ζ or CD3z) chains, which together transduce activation signals into the T cell interior when the antigen interacts with the TCR heterodimer. The structure of the complete TCR-CD3 complex (i.e., the "TCR signaling complex" or "TCR complex") is shown in [details omitted]. Figure 1As shown in the figure. Various TCR heterodimeric subunits (TCRα or TCRβ) and various CD3 subunits (i.e., CD3γ, CD3δ, CD3ε, and CD3ζ subunits) can be considered as TCR signaling complex subunits (or interchangeable with "TCR subunit"). Detailed descriptions of the TCR and its functional uses are described, for example, in Alcover, A et al., "Cell biology of T cell receptor expression and regulation." Annual Review of Immunology 36 (2018):103-125; Gaud, G. et al., "Regulatory mechanisms in T cell receptor signalling." Nature Reviews Immunology 18.8 (2018):485-497; and Shah, K. et al., "T cell receptor (TCR) signaling in health and disease." Signal Transduction and Targeted Therapy 6.1 (2021):412; each of which is incorporated herein by reference in its entirety.

[0006] TCR signaling plays a crucial role in medicine, including cancer treatment. However, in some cases, TCR signaling may be insufficient to activate T cells. Therefore, there is a need in the art for engineered TCRs with improved activity. Summary of the Invention

[0007] This disclosure provides an engineered T-cell receptor (TCR) complex, referred to as an antigen-specific redirected TCR complex (or “Aspire-TCR”), which substantially comprises an engineered target recognition subunit and / or an exogenous CD3 zeta (i.e., CD3ζ or CD3z) subunit. Such engineered TCR complexes are capable of specifically and efficiently recognizing specific target molecules expressed in target cells (e.g., tumor cells). When expressed in immune cells, the engineered T-cell receptor (TCR) complex can confer specificity and effective cytotoxicity against target cells to immune cells expressing the engineered TCR complex.

[0008] In a first aspect, this disclosure provides an engineered CD3 zeta (CD3z) subunit comprising a co-stimulatory region operatively linked to or incorporated into a CD3 zeta component. The CD3 zeta component comprises mammalian CD3 zeta or a functional portion or variant thereof, and the co-stimulatory region is located within an intracellular domain of the engineered CD3 zeta subunit.

[0009] According to some embodiments, the CD3 zeta component comprises human CD3 zeta or a functional portion or variant thereof, wherein the human CD3 zeta or a functional portion or variant thereof comprises a sequence having at least 80% sequence identity with SEQ ID NO:1.

[0010] According to some implementation schemes, when the engineered CD3 zeta subunit is expressed in immune cells, at least one of the following is satisfied: (1) immune cells expressing the engineered CD3 zeta subunit have reduced activation in the absence of antigen stimulation compared to when immune cells do not express the engineered CD3 zeta subunit; (2) immune cells expressing the engineered CD3 zeta subunit have reduced cytotoxicity against non-target cells compared to when immune cells do not express the engineered CD3 zeta subunit; (3) immune cells expressing the engineered CD3 zeta subunit have increased activation after antigen stimulation compared to when immune cells do not express the engineered CD3 zeta subunit; and (4) immune cells expressing the engineered CD3 zeta subunit have increased immune cell responses against their corresponding target cells compared to when immune cells do not express the engineered CD3 zeta subunit.

[0011] According to some implementations, the CD3 zeta component is a full-length wild-type human CD3 zeta subunit containing an amino acid sequence as shown in SEQ ID NO:1.

[0012] In any embodiment of the engineered CD3 zeta subunit as described above, the co-stimulatory region may comprise a co-stimulatory domain of CD28, 4-1BB, OX40, CD2, CD27, CDS, ICAM-1, LFA-1, or ICOS, or a functional portion or variant thereof. Thus, according to some embodiments of the engineered CD3 zeta subunit, the co-stimulatory region comprises a co-stimulatory domain of CD28, or a functional portion or variant thereof, wherein the co-stimulatory domain of CD28, or a functional portion or variant thereof, comprises a sequence having at least 80% sequence identity with SEQ ID NO:2; however, according to some other embodiments of the engineered CD3 zeta subunit, the co-stimulatory region comprises a co-stimulatory domain of 4-1BB, or a functional portion or variant thereof, wherein the co-stimulatory domain of 4-1BB, or a functional portion or variant thereof, comprises a sequence having at least 80% sequence identity with SEQ ID NO:3.

[0013] According to some embodiments of the engineered CD3 zeta subunit, the co-stimulatory region is operatively fused to the C-terminus of the CD3 zeta assembly. In this context, the fusion between the co-stimulatory region and the CD3 zeta assembly can optionally be performed via a linker (such as a flexible linker), but can optionally be performed directly without any septum sequence or linker in between. According to further embodiments of the engineered CD3 zeta subunit, the co-stimulatory region is located between the transmembrane (TM) domain and the intracellular domain (ICD) of the CD3 zeta assembly (i.e., the co-stimulatory region is inserted in the region between the TM and ICD of the CD3 zeta subunit).

[0014] According to some implementation schemes, the engineered CD3 zeta subunit further includes a STAT-binding region within its intracellular domain.

[0015] In some embodiments herein, the STAT binding region may include a STAT3 binding motif comprising at least 50%, 75%, or 100% of the amino acid sequence identical to that of SEQ ID NO:22, and further optionally, the STAT3 binding motif comprises at least 50%, 75%, or 100% of the amino acid sequence identical to that of SEQ ID NO:21; in still other embodiments, the STAT binding region may include a STAT5 binding motif comprising at least 50%, 75%, or 100% of the amino acid sequence identical to that of SEQ ID NO:23, and further optionally, the STAT5 binding motif comprises at least 50%, 75%, or 100% of the amino acid sequence identical to that of SEQ ID NO:24.

[0016] In this document, the STAT binding region is optionally located in the region of the CD3 zeta component corresponding to positions 150-164 of SEQ ID NO:1, and according to some embodiments, the STAT binding region is an insertion at a position between positions 157 and 158 of the CD3 zeta component corresponding to SEQ ID NO:1. Therefore, according to some embodiments in which a STAT3 binding motif is inserted, the CD3 zeta component comprises at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:20; however, according to some other embodiments in which a STAT5 binding motif is inserted, the CD3 zeta component comprises at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:29.

[0017] According to any of the above embodiments in which the engineered CD3 zeta subunit contains a STAT-binding region within its intracellular domain, when the engineered CD3 zeta subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the STAT-binding region is not present in the engineered CD3 zeta subunit: (1) the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen; (2) the T cell population exhibits increased potency in vivo against tumors containing target cells; and (3) the T cell population exhibits a higher proportion in vivo in tumor tissues infiltrated by it.

[0018] In any embodiment of the engineered CD3 zeta subunit as described above, the immune cell can be a T lymphocyte (e.g., a T cell), a tumor-infiltrating lymphocyte (TIL), or a natural killer (NK) cell. In some embodiments herein, where the immune cell is a T cell, when the engineered CD3 zeta subunit is expressed in a population of T cells expressing the T cell receptor (TCR), the T cell population exhibits increased surface expression compared to when the co-stimulatory region is not present in the engineered CD3 zeta subunit.

[0019] In connection with the above, a method for modulating the activity of immune cells is further provided. The method substantially includes the step of expressing an engineered CD3 zeta subunit in immune cells according to any of the embodiments described above. By means of the method provided herein, immune cells expressing the engineered CD3 zeta subunit satisfy at least one of the following: (1) the immune cells exhibit reduced activation in the absence of antigen stimulation compared to when the immune cells do not express the engineered CD3 zeta subunit; (2) the immune cells exhibit reduced cytotoxicity against non-target cells compared to when the immune cells do not express the engineered CD3 zeta subunit; (3) the immune cells exhibit increased activation upon antigen stimulation compared to when the immune cells do not express the engineered CD3 zeta subunit; and (4) the immune cells exhibit increased immune cell responses against their corresponding target cells compared to when the immune cells do not express the engineered CD3 zeta subunit.

[0020] In a second aspect, this disclosure further provides an engineered CD3 epsilon (CD3e) subunit comprising a target-recognizing region (i.e., a target-recognizing moiety or target-recognizing portion) and a CD3 epsilon component. The target-recognizing region is operatively fused to an extracellular domain of the engineered CD3epsilon subunit; and the CD3 epsilon component comprises a truncated CD3 epsilon, wherein the truncation is located in a region of the mammalian CD3 epsilon subunit corresponding to its intercellular domain.

[0021] In this document, according to some embodiments of the engineered CD3 epsilon subunit, the CD3 epsilon component comprises a truncated human CD3 epsilon. Optionally, the truncation occurs in the region corresponding to positions 151-179 of SEQ ID NO:5 of the human CD3 epsilon subunit; and therefore, the CD3 epsilon component may optionally comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:10. In this document, it is further configured such that when the engineered CD3 epsilon subunit is expressed in a T cell population, the T cell population exhibits increased surface expression and / or increased amplification of the engineered CD3 epsilon subunit compared to when the CD3 epsilon component comprises a full-length CD3 epsilon subunit.

[0022] According to some embodiments of the engineered CD3 epsilon subunit, the C-terminus of the CD3 epsilon component is the C-terminus of the engineered CD3 epsilon subunit. In this document, it is further configured such that when the engineered CD3 epsilon subunit is expressed in a T cell population, at least one of the following is satisfied: (1) the T cell population exhibits increased surface expression of the engineered CD3 epsilon subunit compared to when the CD3 epsilon component contains a full-length CD3 epsilon subunit; (2) the T cell population contains a higher percentage of central memory T cells compared to when the CD3 epsilon component contains a full-length CD3 epsilon subunit; and (3) the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen compared to when the CD3 epsilon component contains a full-length CD3 epsilon subunit.

[0023] According to some implementation schemes, the engineered CD3 epsilon subunit further includes at least one functional region in its intracellular domain, and the at least one functional region is operatively fused to the C-terminus of the CD3 epsilon component.

[0024] Optionally, at least one functional region herein comprises an immune receptor tyrosine-based activation motif (ITAM), which may be derived from CD3z, CD3g, DAP12, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD28, or CD72. According to some embodiments, the ITAM may comprise: (1) CD3zITAM3, comprising at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:11; or (2) CD3zITAM2-3, comprising at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:12, but may comprise other ITAM sequences known in the art. According to some embodiments, the ITAM is located at (e.g., operably fused to) the C-terminus of an engineered CD3 epsilon subunit. In this document, the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, the ITAM may further comprise a STAT-binding region (e.g., a STAT3-binding motif or a STAT5-binding motif). In this document, it is further configured such that when the engineered CD3 epsilon subunit is expressed in a T cell population, the T cell population contains a higher percentage of central memory T cells compared to when the ITAM is not present in the engineered CD3 epsilon subunit. It should be noted that, optionally, at least one functional region may comprise more than one ITAM, and each ITAM may have the same or different sequences.

[0025] Optionally, at least one functional region comprises any one or a combination of the following: (1) an intracellular domain of FcεR1γ containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:25; (2) an intracellular domain of OX40 containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:26; (3) an intracellular domain of CD40 containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:27; (4) an intracellular domain of DAP12 containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:28; (5) an intracellular domain of 4-1BB containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:28. (6) The amino acid sequence of NO:3 is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:15; (7) The CD40 motif is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:17; (8) The adapter (LAT) motif for activating T cells is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:16; and (9) The CD28 intracellular domain is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:2.

[0026] Optionally, the engineered CD3 epsilon subunit further includes a STAT-binding region within its intracellular domain, the STAT-binding region comprising: (1) a STAT3-binding motif containing at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO:22; or (2) a STAT5-binding motif containing at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO:23. According to some embodiments, the STAT-binding region comprises a STAT3-binding motif containing at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO:21. According to some other embodiments, the STAT-binding region comprises a STAT5-binding motif containing at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO:24.

[0027] Optionally, the engineered CD3 epsilon subunit further includes ITAM in its intracellular domain, and ITAM includes a STAT-binding region. According to some embodiments, ITAM is a CD3z ITAM2-3 motif containing at least 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:18. According to some other embodiments, ITAM is a CD3z ITAM3 motif containing at least 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:19.

[0028] According to some embodiments of the engineered CD3 epsilon subunit, at least one functional region comprises both an intracellular domain of FcεR1γ and an intracellular domain of OX40. Optionally, at least one functional region comprises a first complex functional region comprising both the intracellular domain of FcεR1γ and the intracellular domain of OX40 in the direction from the N-terminus to the C-terminus, and the first complex functional region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:13. Further optionally, at least one functional region further comprises ITAM (e.g., CD3z ITAM3, or CD3z ITAM2-3, etc.) at the C-terminus of the first complex functional region. Optionally in this study, when the engineered CD3 zeta subunit was expressed in a T cell population, the T cell population exhibited increased potency in vivo against tumors containing target cells corresponding to the T cell population compared to when the FcεR1γ intracellular domain, OX40 intracellular domain, and ITAM were not present in the engineered CD3 zeta subunit.

[0029] According to some embodiments of the engineered CD3 epsilon subunit, at least one functional region comprises both a CD40 intracellular domain and a DAP12 intracellular domain. Optionally, at least one functional region comprises a second complex functional region comprising both the CD40 intracellular domain and the DAP12 intracellular domain from the N-terminus to the C-terminus, and the second complex functional region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:14. Further optionally, at least one functional region further comprises ITAM (e.g., CD3z ITAM3, or CD3z ITAM2-3, etc.) at the C-terminus of the second complex functional region.

[0030] In any of the embodiments described above, the engineered CD3 epsilon subunit further comprises a 4-1BB intracellular domain and an ITAM (e.g., CD3z ITAM3, or CD3z ITAM2-3, etc.) from its N-terminus to its C-terminus. In this document, when the engineered CD3 epsilon subunit is expressed in T cells specifically targeting cells with a specific antigen, at least one of the following is satisfied compared to when the 4-1BB intracellular domain and ITAM are not present in the engineered CD3 epsilon subunit: (1) the T cells exhibit increased cytotoxicity against the target cells in vitro; and (2) the T cells exhibit increased antigen-responsiveness in vitro.

[0031] According to some embodiments of the engineered CD3 epsilon subunit, at least one functional region contains both a 4-1BB motif and a LAT motif. Further optionally, the engineered CD3 epsilon subunit further contains an ITAM (e.g., CD3z ITAM3, or CD3z ITAM2-3, etc.) at its C-terminus. Optionally herein, when the engineered CD3 epsilon subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the 4-1BB motif, LAT motif, and ITAM are not present in the engineered CD3epsilon subunit: (1) the T cell population contains a higher percentage of central memory T cells; and (2) the T cell population exhibits increased in vitro expansion upon repeated stimulation with the antigen. Further optionally, the ITAM contains a STAT-binding region (e.g., a STAT3-binding motif, or a STAT5-binding motif, etc.).

[0032] According to some embodiments of the engineered CD3 epsilon subunit, at least one functional region contains both the CD40 motif and the LAT motif. Further optionally, the engineered CD3 epsilon subunit further contains ITAM (e.g., CD3z ITAM3, or CD3z ITAM2-3, etc.) at its C-terminus. Optionally, when the engineered CD3 zeta subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the CD40 motif, LAT motif, and ITAM are not present in the engineered CD3 zeta subunit: (1) the T cell population exhibits increased surface expression of the engineered CD3 epsilon subunit; (2) the T cell population contains a higher percentage of naive T cells. (3) The T cell population contains a higher percentage of central memory T cells; (4) The T cell population exhibits increased potency against tumors containing target cells in vivo; and (5) The T cell population exhibits a higher proportion in tumor tissues infiltrated by it in vivo.

[0033] In any implementation of the engineered CD3 epsilon subunit as described above, the target recognition region may optionally be operatively incorporated into the CD3 epsilon component.

[0034] In any embodiment of the engineered CD3 epsilon subunit as described above, the target recognition region may include: (1) an antigen-binding region; or (2) a ligand or fragment thereof that binds to a cell surface receptor expressed on a target cell of an immune cell. According to some implementation schemes, the target recognition region includes an antigen-binding region, which optionally includes a single-stranded variable fragment (scFv) that specifically recognizes ALPP, LYPD3, IL13Ra2, BCMA, CD16, CD19, CD20, CD22, CD27, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, mesothelin, NKG2D, phosphatidylinositol proteoglycan-3 (GPC-3), FAP, FRa (folate receptor α), EGFR, EGFR vIII, IL-11Ra (IL11 receptor α), VEGFR-II, B7-H6, and DNAM-1. In some embodiments, the antigen-binding region comprises a single-chain variable fragment (scFv) targeting ALPP. According to some other embodiments, the target recognition region comprises an antigen-binding region, which may optionally comprise a single-domain antibody (sdAb or nanobody). According to some embodiments, the target recognition region may comprise a ligand or a fragment thereof, and the ligand may optionally be IL13 (E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRIN B2, CTLX, LFA-1, or FSH. In some embodiments, the ligand is IL13 (E13Y).

[0035] According to some embodiments of the engineered CD3 epsilon subunit, a flexible connector may be located between the target recognition region and the CD3epsilon component, and may contain the sequence of SEQ ID NO:6.

[0036] In a third aspect, this disclosure further provides an engineered TCR complex system.

[0037] According to some embodiments, the engineered TCR complex system comprises an engineered CD3 epsilon subunit according to any embodiment of the engineered CD3 epsilon subunit as described above in the second aspect. Optionally, the engineered TCR complex system may further comprise an engineered CD3 zeta subunit according to any embodiment of the engineered CD3 zeta subunit as described above in the first aspect.

[0038] In some embodiments of the engineered TCR complex system provided herein, the co-stimulatory region of the engineered CD3 zeta subunit comprises: (1) a co-stimulatory domain of CD28, or a functional portion or functional variant thereof; or (2) a co-stimulatory domain of 4-1BB, or a functional portion or functional variant thereof.

[0039] In embodiments of an engineered TCR complex system comprising both an engineered CD3 epsilon subunit and an engineered CD3 zeta subunit, one or both of the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit may optionally include a STAT-binding region within their intracellular domain.

[0040] In embodiments of an engineered TCR complex system in which both the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit contain STAT-binding regions within their intracellular domains, the STAT-binding region of the engineered CD3 epsilon subunit may optionally be the same as or different from the STAT-binding region of the engineered CD3 zeta subunit. In some embodiments in which the STAT-binding regions of the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit are different, the STAT-binding regions of the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit are (1) STAT3-binding motifs and STAT5-binding motifs, respectively, or (2) STAT5-binding motifs and STAT3-binding motifs, respectively.

[0041] In an embodiment of an engineered TCR complex system in which only the engineered CD3 zeta subunit contains a STAT-binding region within its intracellular domain, the STAT-binding region contains a STAT3-binding motif or a STAT5-binding motif.

[0042] In any embodiment of the engineered TCR complex system described above, when the engineered CD3 epsilon subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the STAT-binding region is not present in either the engineered CD3 epsilon subunit or the engineered CD3 zeta subunit: (1) the T cell population contains a higher percentage of terminally differentiated effector T cells; (2) the T cell population exhibits increased antigen-specific stimulation in vitro; and (3) the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen.

[0043] According to some embodiments, the engineered TCR complex system comprises an engineered CD3 zeta subunit according to any embodiment of the engineered CD3 zeta subunit as described above in the first aspect, and the engineered TCR complex system further comprises an engineered target-recognition TCR subunit. Hereinafter, the engineered target-recognition TCR subunit may be based on one of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, or CD3δ, and preferably may be based on one of TCRα, TCRβ, CD3ε, CD3γ, or CD3δ. Optionally hereafter, the engineered target-recognition TCR subunit comprises a target-recognition region or target-recognition portion, which may comprise (1) an antigen-binding region (e.g., a single-chain variable fragment (scFv) or a single-domain antibody); or (2) a ligand or fragment thereof that binds to a cell surface receptor expressed on a target cell of an immune cell.

[0044] In a fourth aspect, this disclosure further provides a chimeric polypeptide comprising a target recognition region or target recognition portion operatively linked to or incorporated into one of the TCR alpha subunit, TCR beta subunit, CD3 gamma subunit, CD3 delta subunit, or CD3 epsilon subunit, or a functional portion or functional variant thereof. The target recognition region or target recognition portion comprises a ligand, its functional portion, or a functional variant thereof. Herein, when the chimeric polypeptide is expressed in immune cells, the target recognition region of the chimeric polypeptide is capable of binding to a cell surface receptor expressed on a target cell of the immune cell. Therefore, this fourth aspect substantially provides an engineered target-recognizing TCR subunit based on any one of TCRα, TCRβ, CD3ε, CD3γ, or CD3δ, wherein the target recognition region comprises a ligand portion that enables the TCR subunit, when expressed in immune cells (e.g., T cells), to specifically recognize and bind to a homologous ligand receptor expressed on a target cell.

[0045] According to some embodiments of the chimeric peptide, the target recognition region is operatively linked to or incorporated into the CD3 epsilon subunit or a functional portion or variant thereof.

[0046] In this document, according to some embodiments, the CD3 epsilon subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:5; according to some other embodiments, the CD3epsilon subunit comprises a truncated CD3 epsilon that comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0047] Optionally, the ligand is selected from IL13 (E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRIN B2, CTLX, LFA-1, or FSH.

[0048] According to some embodiments, the ligand is IL13(E13Y), which may optionally contain at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:4.

[0049] In a fifth aspect, this disclosure further provides engineered immune cells comprising an engineered TCR complex system according to any embodiment described in the third aspect above, or a chimeric polypeptide according to any embodiment described in the fourth aspect above. Optionally, the engineered immune cells may be T lymphocytes (or T cells), tumor-infiltrating lymphocytes (TILs), or natural killer (NK) cells.

[0050] In a sixth aspect, this disclosure further provides a method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of engineered immune cells according to any of the embodiments described in the fifth aspect above. In this document, the subject may be a human, but may also be a mammal, such as a dog, cat, or monkey.

[0051] As used elsewhere in this document and throughout the disclosure, “a / an / a type (a)” or “an / an / a type (an)” means “at least one / an / a type” or “more than one / an / a type”. In one instance, in the engineered CD3 zeta (CD3z) subunit provided above in the first aspect, which includes “co-stimulatory regions” operatively connected to or incorporated into the CD3 zeta component, the phrase “co-stimulatory region” can be interpreted as meaning “at least one co-stimulatory region”.

[0052] In one aspect, this disclosure relates to an engineered CD3 zeta (CD3z) subunit comprising a co-stimulatory region operatively linked to or incorporated into a CD3 zeta component, wherein in some embodiments the CD3 zeta component comprises human CD3 zeta or a functional portion or variant thereof, said human CD3 zeta or a functional portion or variant thereof comprising a sequence having at least 80% sequence identity with SEQ ID NO:1; and the co-stimulatory region is located within an intracellular domain of the engineered CD3 zeta subunit. In some embodiments, when expressed in immune cells, at least one of the following is satisfied: (1) immune cells expressing the engineered CD3 zeta subunit exhibit reduced activation in the absence of antigen stimulation compared to immune cells not expressing the engineered CD3 zeta subunit; (2) immune cells expressing the engineered CD3 zeta subunit exhibit reduced cytotoxicity against non-target cells compared to immune cells not expressing the engineered CD3 zeta subunit; (3) immune cells expressing the engineered CD3 zeta subunit exhibit increased activation upon antigen stimulation compared to immune cells not expressing the engineered CD3 zeta subunit; and (4) immune cells expressing the engineered CD3 zeta subunit exhibit increased immune cell responses against their corresponding target cells compared to immune cells not expressing the engineered CD3 zeta subunit. In some embodiments, the CD3 zeta component comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the co-stimulatory region comprises a co-stimulatory domain, or a functional portion or variant thereof, of a protein selected from the group consisting of CD28, 4-1BB, OX40, CD2, CD27, CDS, ICAM-1, LFA-1, and ICOS. In some embodiments, the co-stimulatory region comprises a co-stimulatory domain, or a functional portion or variant thereof, of CD28, wherein the co-stimulatory domain, or functional portion or variant thereof, comprises a sequence having at least 80% sequence identity with SEQ ID NO:2. In some embodiments, the co-stimulatory region comprises a co-stimulatory domain, or a functional portion or variant thereof, of 4-1BB, wherein the co-stimulatory domain, or functional portion or variant thereof, comprises a sequence having at least 80% sequence identity with SEQ ID NO:3. In some embodiments, the co-stimulatory region is operatively fused to the C-terminus of the CD3 zeta assembly. In some embodiments, the co-stimulatory region is located between the transmembrane domain and the intracellular domain of the CD3 zeta assembly.

[0053] In some embodiments, the engineered CD3 zeta subunit described herein further includes a STAT-binding region within its intracellular domain. In some embodiments, the STAT-binding region includes a STAT3-binding motif comprising at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the STAT3-binding motif comprises at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the STAT-binding region includes a STAT5-binding motif comprising at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the STAT5-binding motif comprises at least 50%, 75%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the STAT-binding region is located within the region of the CD3 zeta component corresponding to positions 150-164 of SEQ ID NO: 1. In some embodiments, the STAT binding region is an insertion at the position between positions 157 and 158 of the CD3 zeta component corresponding to SEQ ID NO:1. In some embodiments, the CD3 zeta component comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:20. In some embodiments, the CD3 zeta component comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:29.

[0054] In some implementations, when the engineered CD3 zeta subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the STAT-binding region is not present in the engineered CD3 zeta subunit: the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen; the T cell population exhibits increased potency against tumors containing target cells in vivo; and the T cell population exhibits a higher proportion in tumor tissue infiltrated by it in vivo.

[0055] In some embodiments, the immune cells are T lymphocytes, tumor-infiltrating lymphocytes (TILs), or natural killer (NK) cells. In some embodiments, when the engineered CD3 zeta subunit is expressed in a population of T cells expressing the T cell receptor (TCR), the T cell population exhibits increased surface expression compared to when the co-stimulatory region is not present in the engineered CD3 zeta subunit.

[0056] In one aspect, this disclosure relates to a method for modulating the activity of immune cells, comprising: expressing the engineered CD3 zeta subunit described herein in immune cells. In some embodiments, immune cells expressing the engineered CD3 zeta subunit: (1) exhibit reduced activation in the absence of antigen stimulation compared to when immune cells do not express the engineered CD3 zeta subunit; (2) exhibit reduced cytotoxicity against non-target cells compared to when immune cells do not express the engineered CD3 zeta subunit; (3) exhibit increased activation upon antigen stimulation compared to when immune cells do not express the engineered CD3 zeta subunit; or (4) exhibit an increased immune cell response against corresponding target cells compared to when immune cells do not express the engineered CD3 zeta subunit.

[0057] In one aspect, this disclosure relates to an engineered CD3 epsilon (CD3e) subunit comprising a target recognition region and a CD3epsilon component, wherein in some embodiments, the target recognition region is operatively fused to an extracellular domain of the engineered CD3 epsilon subunit; and the CD3 epsilon component comprises a truncated CD3 epsilon, wherein the truncation is located at a region of the human CD3epsilon subunit corresponding to its intercellular domain. In some embodiments, the truncation is located in the region of the human CD3 epsilon subunit corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the CD3 epsilon component comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:10. In some embodiments, the CD3 epsilon component comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, when the engineered CD3 epsilon subunit is expressed in a T cell population, the T cell population exhibits increased surface expression and / or increased expansion of the engineered CD3 epsilon subunit compared to when the CD3 epsilon component contains a full-length CD3 epsilon subunit. In some embodiments, the C-terminus of the CD3 epsilon component is the C-terminus of the engineered CD3epsilon subunit. In some embodiments, when the engineered CD3 epsilon subunit is expressed in a T cell population, at least one of the following is satisfied: (1) the T cell population exhibits increased surface expression of the engineered CD3 epsilon subunit compared to when the CD3 epsilon component contains a full-length CD3 epsilon subunit; (2) the T cell population contains a higher percentage of central memory T cells compared to when the CD3 epsilon component contains a full-length CD3epsilon subunit; and (3) the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen compared to when the CD3epsilon component contains a full-length CD3 epsilon subunit.

[0058] In some embodiments, the engineered CD3 epsilon subunit described herein further includes at least one functional region within its intracellular domain, and in some embodiments, at least one functional region is operatively fused to the C-terminus of the CD3 epsilon component.

[0059] In some embodiments, at least one functional region comprises an immune receptor tyrosine-based activation motif (ITAM). In some embodiments, the ITAM is derived from CD3z, CD3g, DAP12, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD28, or CD72. In some embodiments, the ITAM comprises: (1) CD3z ITAM3, which comprises at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:11; or (2) CD3z ITAM2-3, which comprises at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:12. In some embodiments, the ITAM is located at the C-terminus of an engineered CD3 epsilon subunit. In some embodiments, the ITAM is operatively fused to the C-terminus of the CD3 epsilon component; in some embodiments, the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, the ITAM comprises a STAT binding region; in some embodiments, the STAT binding region is a STAT3 binding motif or a STAT5 binding motif.

[0060] In some implementations, when the engineered CD3 epsilon subunit is expressed in the T cell population, the T cell population contains a higher percentage of central memory T cells compared to when ITAM is not present in the engineered CD3 epsilon subunit.

[0061] In some embodiments, at least one functional region comprises any or a combination of the following: (1) an intracellular domain of FcεR1γ containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:25; (2) an intracellular domain of OX40 containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:26; (3) an intracellular domain of CD40 containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:27; (4) an intracellular domain of DAP12 containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:28; and (5) an intracellular domain of 4-1BB containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:28. (6) The amino acid sequence of NO:3 is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:15; (7) The CD40 motif is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:17; (8) The adapter (LAT) motif for activating T cells is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:16; and (9) The CD28 intracellular domain is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:2.

[0062] In some embodiments, the engineered CD3 epsilon subunit described herein further includes a STAT-binding region within its intracellular domain. In some embodiments, the STAT-binding region includes: (1) a STAT3 binding motif containing at least 50%, 75%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:22; or (2) a STAT5 binding motif containing at least 50%, 75%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:23. In some embodiments, the STAT-binding region includes a STAT3 binding motif containing at least 50%, 75%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:21. In some embodiments, the STAT-binding region includes a STAT5 binding motif containing at least 50%, 75%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:24. In some embodiments, the engineered CD3 epsilon subunit described herein further includes an ITAM in its intracellular domain, and in some embodiments, the ITAM includes a STAT-binding region. In some embodiments, the ITAM is a CD3z ITAM2-3 motif containing at least 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the ITAM is a CD3z ITAM3 motif containing at least 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 19. In some embodiments, at least one functional region includes both an FcεR1γ intracellular domain and an OX40 intracellular domain. In some embodiments, at least one functional region includes a first complex functional region comprising an FcεR1γ intracellular domain and an OX40 intracellular domain from the N-terminus to the C-terminus. In some embodiments, the first complex functional region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:13. In some embodiments, at least one functional region further comprises ITAM at the C-terminus of the first complex functional region. In some embodiments, the ITAM comprises CD3zITAM3 or CD3zITAM2-3.

[0063] In some implementations, when the engineered CD3 zeta subunit is expressed in a T cell population, the T cell population exhibits increased potency against tumors containing target cells in vivo compared to when the FcεR1γ intracellular domain, OX40 intracellular domain, and ITAM are not present in the engineered CD3 zeta subunit.

[0064] In some embodiments, at least one functional region comprises both a CD40 intracellular domain and a DAP12 intracellular domain. In some embodiments, at least one functional region comprises a second complex functional region comprising both a CD40 intracellular domain and a DAP12 intracellular domain from the N-terminus to the C-terminus. In some embodiments, the second complex functional region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, at least one functional region further comprises ITAM at the C-terminus of the second complex functional region. In some embodiments, the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3.

[0065] In some embodiments, the engineered CD3 epsilon subunit described herein further comprises a 4-1BB intracellular domain and an ITAM from its N-terminus to its C-terminus, wherein in some embodiments, the ITAM comprises CD3zITAM3 or CD3zITAM2-3. In some embodiments, when the engineered CD3 epsilon subunit is expressed in T cells specifically targeting cells with a specific antigen, at least one of the following is satisfied compared to when the 4-1BB intracellular domain and ITAM are not present in the engineered CD3epsilon subunit: the T cells exhibit increased cytotoxicity against the target cells in vitro; and the T cells exhibit increased antigen-responsiveness in vitro.

[0066] In some embodiments, at least one functional region contains both the 4-1BB motif and the LAT motif. In some embodiments, the engineered CD3 epsilon subunit described herein further includes ITAM at the C-terminus of the engineered CD3 epsilon subunit; in some embodiments, the ITAM contains CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, when the engineered CD3 epsilon subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the 4-1BB motif, LAT motif, and ITAM are not present in the engineered CD3 epsilon subunit: the T cell population contains a higher percentage of central memory T cells; and the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen. In some embodiments, the ITAM contains a STAT-binding region; in some embodiments, the STAT-binding region is a STAT3-binding motif or a STAT5-binding motif.

[0067] In some embodiments, at least one functional region contains both the CD40 motif and the LAT motif. In some embodiments, the engineered CD3 epsilon subunit described herein further includes ITAM at the C-terminus of the engineered CD3 epsilon subunit; in some embodiments, the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, when the engineered CD3 zeta subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the CD40 motif, LAT motif, and ITAM are not present in the engineered CD3 zeta subunit: the T cell population exhibits increased surface expression of the engineered CD3 epsilon subunit; the T cell population contains a higher percentage of naive T cells; the T cell population contains a higher percentage of central memory T cells; the T cell population exhibits increased potency against tumors containing target cells in vivo; and the T cell population exhibits a higher proportion in tumor tissue infiltrated by it in vivo.

[0068] In some embodiments, the target recognition region is operatively incorporated into the CD3 epsilon component. In some embodiments, the target recognition region comprises: (1) an antigen-binding region; or (2) a ligand or fragment thereof that binds to a cell surface receptor expressed on a target cell of an immune cell. In some embodiments, the antigen-binding region comprises a single-stranded variable fragment (scFv) that specifically recognizes ALPP, LYPD3, IL13Ra2, BCMA, CD16, CD19, CD20, CD22, CD27, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, mesothelin, NKG2D, phosphatidylinositol proteoglycan-3 (GPC-3), FAP, FRa (folate receptor α), EGFR, EGFR vIII, IL-11Ra (IL11 receptor α), VEGFR-II, B7-H6, and DNAM-1. In some embodiments, the antigen-binding region comprises a single-stranded variable fragment (scFv) targeting ALPP. In some embodiments, the antigen-binding region comprises a single-domain antibody (sdAb or nanobody). In some embodiments, the ligand is selected from the group consisting of: IL13 (E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRIN B2, CTLX, LFA-1, and FSH. In some embodiments, the ligand is IL13 (E13Y).

[0069] In some embodiments, the engineered CD3 epsilon subunit described herein further includes a flexible connector between the target recognition region and the CD3epsilon component. In some embodiments, the flexible connector includes the sequence of SEQ ID NO:6.

[0070] In one aspect, this disclosure relates to an engineered TCR complex system comprising at least one of the following: the engineered CD3 zeta subunit described herein; and the engineered CD3 epsilon subunit described herein. In some embodiments, the engineered TCR complex system described herein comprises both the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit described herein.

[0071] In some embodiments, the co-stimulatory region of the engineered CD3 zeta subunit includes: a co-stimulatory domain of CD28, or a functional portion or variant thereof; or a co-stimulatory domain of 4-1BB, or a functional portion or variant thereof. In some embodiments, one or both of the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit include a STAT-binding region within their intracellular domain. In some embodiments, both the engineered CD3 epsilon subunit and the engineered CD3 zeta subunit include a STAT-binding region within their intracellular domain, and in some embodiments, the STAT-binding region of the engineered CD3 epsilon subunit is different from the STAT-binding region of the engineered CD3 zeta subunit. In some embodiments, the STAT-binding region of the engineered CD3 epsilon subunit and the STAT-binding region of the engineered CD3 zeta subunit (1) are a STAT3-binding motif and a STAT5-binding motif, respectively, or (2) are a STAT5-binding motif and a STAT3-binding motif, respectively. In some embodiments, only the engineered CD3 zeta subunit contains a STAT-binding region within its intracellular domain; in some embodiments, the STAT-binding region contains a STAT3-binding motif or a STAT5-binding motif. In some embodiments, when the engineered CD3 epsilon subunit is expressed in a T cell population, at least one of the following is met compared to when the STAT-binding region is not present in either the engineered CD3 epsilon subunit or the engineered CD3 zeta subunit: the T cell population contains a higher percentage of terminally differentiated effector T cells; the T cell population exhibits increased antigen-specific stimulation in vitro; and the T cell population exhibits increased in vitro expansion after repeated stimulation with the antigen. In some embodiments, the engineered TCR complex system described herein comprises an engineered CD3 zeta subunit and further comprises an engineered target-recognition TCR subunit. In some embodiments, the TCR subunit is based on one of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, or CD3δ, and preferably on one of TCRα, TCRβ, CD3ε, CD3γ, or CD3δ. In some embodiments, the engineered target-recognition TCR subunit comprises a target recognition region comprising: (1) an antigen-binding region comprising at least one of a single-chain variable fragment (scFv) or a single-domain antibody; or (2) a ligand or fragment thereof that binds to a cell surface receptor expressed on a target cell of an immune cell.

[0072] In one aspect, this disclosure relates to a chimeric polypeptide comprising a target recognition region operatively linked to or incorporated into one of the TCR alpha subunit, TCR beta subunit, CD3 gamma subunit, CD3 delta subunit, or CD3 epsilon subunit, or a functional portion or variant thereof. In some embodiments, the target recognition region comprises a ligand, a functional portion thereof, or a functional variant thereof. In some embodiments, when the chimeric polypeptide is expressed in immune cells, the target recognition region of the chimeric polypeptide is capable of binding to a cell surface receptor expressed on a target cell of the immune cell. In some embodiments, the target recognition region is operatively linked to or incorporated into a CD3 epsilon subunit, or a functional portion or variant thereof. In some embodiments, the CD3 epsilon subunit comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the CD3 epsilon subunit comprises a truncated CD3epsilon, said truncated CD3 epsilon comprising at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:10. In some embodiments, the ligand is selected from the group consisting of IL13(E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRIN B2, CTLX, LFA-1, and FSH. In some embodiments, the ligand is IL13(E13Y). In some embodiments, the IL13(E13Y) ligand comprises at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:4.

[0073] In one aspect, this disclosure relates to engineered immune cells comprising: the engineered TCR complex system described herein; and / or the chimeric polypeptide described herein. In some embodiments, the engineered immune cells are T lymphocytes, tumor-infiltrating lymphocytes (TILs), or natural killer (NK) cells.

[0074] In one aspect, this disclosure relates to a method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of the engineered immune cells described herein.

[0075] As used herein, the term "immune cell" may mean any of the following: T lymphocytes (including αβT cells or γδT cells), tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, or NK T cells, or any of these cells that have been engineered (e.g., cells expressing TCRs or chimeric antigen receptors (CARs)). In the examples provided in this disclosure, T lymphocytes or T cells are used as illustrative but not limiting examples of immune cells.

[0076] As used herein, a “single-chain variable fragment” or “scFv” antibody fragment contains the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Typically, scFv polypeptides further include a polypeptide linker between the VH and VL domains, allowing scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies. Springer Berlin Heidelberg, 1994, pp. 269-315.

[0077] As used herein, the term "TCR signal transduction complex" or "TCR complex" refers to a complex formed between a TCR subunit (preferably TCRα and TCRβ, but may also include TCRδ and TCRγ) and a CD3 complex subunit (CD3ε, CD3γ, CD3δ, and CD3ζ); the term "TCR signal transduction complex subunit" or "TCR subunit" as used herein refers to a subunit of the TCR complex, including, for example, TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ. One or more of the TCR subunits may be engineered TCR subunits provided in this disclosure.

[0078] As used herein, the term "STAT binding region" includes, but is not limited to, STAT3 binding motifs (e.g., SEQ ID NO:22) and / or STAT5 binding motifs (e.g., SEQ ID NO:23). In some embodiments, the STAT binding region comprises one STAT3 or STAT5 binding motif. In some embodiments, the STAT binding region comprises two or more STAT3 or STAT5 binding motifs.

[0079] As used herein, the term “immunoreceptor-based tyrosine activation motif (ITAM)” refers to a conserved four-amino acid sequence that is repeated twice and is present in the cytoplasmic tail (i.e., endodomain) of certain cell surface proteins of the immune system. Details of ITAM can be found, for example, Love, PE et al., “ITAM-mediated signaling by the T-cell antigen receptor.” Cold Spring Harbor Perspectives in Biology 2.6 (2010):a002485, which is incorporated herein by reference in its entirety.

[0080] As used herein, the term "amplification capacity" refers to the ability of immune cells (e.g., immune cells expressing any engineered TCR complex described herein) to proliferate (e.g., approximately 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, or 30 days after transfection). In some embodiments, "increased amplification capacity" means an increase in the rate of proliferation of at least 1.1-fold (i.e., 110% or more) relative to a reference immune cell that has not been transduced to express the corresponding subunit.

[0081] As used herein, the term "cytolytic toxicity" refers to the ability of immune cells to kill specific target cells when they are co-cultured with them. As used herein, the term "specific target cell" refers to a cell expressing an antigen that can be specifically recognized by a CAR or TCR (e.g., Aspire-TCR) on an immune cell. In some embodiments, "increased cytolytic toxicity" means an increase in the killing efficiency of specific target cells of not less than 1.1-fold (i.e., 110% or more) compared to the use of control immune cells (e.g., untransduced immune cells).

[0082] As used herein, the term "Aspire-TCR" can refer to any Aspire-TCR construct described herein.

[0083] As used herein, the terms “increase,” “increase,” “decrease,” or “reduction,” “change,” “alter,” “variation,” “higher,” “lower,” or similar terms refer to a change in level of not less than 10% compared to a reference level.

[0084] As used in this paper, the phrase “substantially unchanged” means that the level of the tested variability (such as the expression level of the target CAR / TCR) changes by less than 5% when comparing a later time point to an earlier reference time point. If the change is greater than or equal to 5%, such a change can be considered as an “increase” or “decrease” as referred to in this paper.

[0085] As used herein, the phrase “background activation” or “basal activation” refers to the level of activation of immune cells in the absence of a corresponding target cell. In some embodiments, the target cell may express one or more types of target molecules that can be specifically recognized by target recognition regions expressed by the immune cell (e.g., any target recognition regions described herein).

[0086] As used in this article, the phrase “target cell” refers to a cell that can be specifically targeted or killed by immune cells.

[0087] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are considered identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of gaps introduced for optimal alignment and the length of each gap. For example, the Blossum 62 scoring matrix can be used to perform sequence comparison and determine the percentage identity between two sequences with the following parameters: a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail.

[0089] Other features and advantages of the invention will be apparent from the following detailed description and drawings, as well as from the claims. Attached Figure Description

[0090] Figure 1 The structure of a conventional TCR signal transduction complex is shown.

[0091] Figure 2A-2C The engineered CD3 zeta subunits according to some embodiments of this disclosure are shown respectively. Figure 2A ), engineered CD3 epsilon subunit ( Figure 2B ), and ligand-based target recognition of Aspire-TCR subunits ( Figure 2C (Structure diagram of )

[0092] Figure 3 The structural diagrams of the four Aspire-TCR constructs involved in the experiment are shown.

[0093] Figures 4A-4E It shows NT cells ( Figure 4A ), CD3e ( Figure 4B ), BBe( Figure 4C ), CD3e-28z ( Figure 4D ), and CD3e-BBz ( Figure 4E IL13 expression in different Aspire-T cells, including )

[0094] Figure 5 The study demonstrated the specific cytotoxicity of IL13(E13Y)Aspire-T cells against the target tumor cells (U251 MG glioma cell line).

[0095] Figure 6 The study showed that activated IL13(E13Y) Aspire-T cells produce IFN-γ in response to tumor cells.

[0096] Figure 7A The expression level of IL13Ra1 in THP1 cells was shown.

[0097] Figure 7B The expression level of IL13Ra2 in THP1 cells was shown.

[0098] Figure 7C The study demonstrated the cytotoxicity of IL13(E13Y)Aspire-T cells against non-target THP-1 cells.

[0099] Figure 8A The percentage of cells producing IFN-γ is shown in the presence of THP1 cells (“THP-1”) or in the absence of THP1 cells (“no target”). Activation of IL13 (E13Y) Aspire-T cells against non-target THP-1 cells was evaluated.

[0100] Figure 8BThe study showed the IFN-γ secretion level of activated IL13(E13Y)Aspire-T cells in the presence of non-target THP-1 cells.

[0101] Figure 9 The structural diagrams of two anti-ALPP Aspire-TCR constructs are shown.

[0102] Figures 10A-10C The image shows non-transduced T cells (“NT”). Figure 10A ), F8-BBe Aspire-T cells ( Figure 10B ), and F8-28z Aspire-T cells ( Figure 10C The expression of F8 scFv in different cells, including )

[0103] Figure 11A-11B The results showed that F8 Aspire-T cells were effective against SiHa cells ( Figure 11A ) and Caski cells ( Figure 11B Specific cytotoxicity of target tumor cells, including those containing tumor cells.

[0104] Figure 12 The study showed the IFN-γ secretion level of activated F8 Aspire-T cells in the presence of target tumor cells.

[0105] Figure 13A The expression level of ALPP ligand in A549 cells was shown.

[0106] Figure 13B The expression level of ALPP ligands in SiHa cells was shown.

[0107] Figure 13C The study demonstrated the background cytotoxicity of F8 Aspire-T cells against non-target A549 cells.

[0108] Figure 14 Various DNA constructs encoding Aspire-TCR for designing AK are shown.

[0109] Figure 15A-15K The structures of the Aspire-TCR complex designed for AK are shown, once expressed in T cells.

[0110] Figures 16A-16D It showed the difference between the control NT T cells ( Figure 16A Compared to other Aspire-T cells (including those expressing scFv-CD3e-28z), different Aspire-T cells (including those expressing scFv-CD3e-28z) Figure 16B ), scFv-ΔCD3e-28z ( Figure 16C ), or scFv-ΔCD3eZ-28z ( Figure 16DscFv expression between Aspire-T cells.

[0111] Figure 17A-17F It showed the difference between the control NT T cells ( Figure 17A ) and expression scFv-CD3e-28z ( Figure 17B ) or scFv-ΔCD3e-28z( Figure 17C Compared to Aspire-T cells expressing scFv-ΔCD3e-FOZ-28z, different Aspire-T cells (including those expressing scFv-ΔCD3e-FOZ-28z) Figure 17D ), scFv-ΔCD3e-40DZ-28z Figure 17E ), or scFv-ΔCD3e-41BBZ-28z ( Figure 17F scFv expression between Aspire-T cells.

[0112] Figures 18A-18F This showed the effect of Aspire-T cells expressing scFv-ΔCD3e-28z ( Figure 18A Compared to other Aspire-T cells (including those expressing scFv-ΔCD3e-41BBM-M1Z-28z), different Aspire-T cells (including those expressing scFv-ΔCD3e-41BBM-M1Z-28z) Figure 18B ), scFv-ΔCD3e-41BBM-M1Z-28z( Figure 18C ), scFv-ΔCD3e-28z-S3( Figure 18D ), scFv-ΔCD3e-S5-28z-S3( Figure 18E ), or scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 ( Figure 18F scFv expression between Aspire-T cells.

[0113] Figures 19A-19C The expansion of scFv Aspire-T cells in vitro was demonstrated.

[0114] Figures 20A-20B The proportion of memory T cells in different scFv Aspire-T cells was shown after expansion.

[0115] Figures 21A-21B The study demonstrated the specific cytotoxicity of scFv Aspire-T cells against target SiHa tumor cells.

[0116] Figures 22A-22B The activation of scFv Aspire-T cells was demonstrated after antigen-specific stimulation of target SiHa tumor cells.

[0117] Figures 23A-23BThis study demonstrates the expansion of CAR+ T cells derived from various scFv Aspire-T cells in response to stimulation by tumor cells.

[0118] Figure 24 The structural diagram of IE06 is shown, where “CD8a” represents the signal peptide from CD8A corresponding to amino acids 1-46 of SEQ ID NO:31, “hIL12p40” represents the human IL12 beta subunit corresponding to amino acids 47-352 of SEQ ID NO:31, “hIL12p35” represents the human IL12 alpha subunit corresponding to amino acids 368-564 of SEQ ID NO:31, and “TM” represents the transmembrane domain corresponding to amino acids 565-610 of SEQ ID NO:31. The linker is located between “hIL12p40” and “hIL12p35”, corresponding to amino acids 353-367 of SEQ ID NO:31.

[0119] Figure 25 The in vivo tumor volume in NSG mice transplanted with tumor cells and treated with armored Aspire-T cells is shown.

[0120] Figure 26 This study demonstrates the in vivo proliferation of IE06-armored scFvAspire-T cells in a tumor cell transplantation model in NSG mice. scFvAspire-T cell proliferation is represented by the hCD45:mCD45 ratio in the peripheral blood of NSG mice. scFvAspire-T cells are generated by transducing human peripheral T cells with various DNA constructs designed to encode different Aspire-TCRs.

[0121] Figure 27 Armored scFv Aspire-T cell infiltration is shown in tumors from NSG mice transplanted with tumor cells. scFv Aspire-T cell infiltration is represented by the hCD45:mCD45 ratio in the tumor tissue.

[0122] Figure 28 The relevant amino acid sequences discussed in this disclosure are listed. Detailed Implementation

[0123] This disclosure provides an engineered T-cell receptor (TCR) signaling complex, referred to as an “antigen-specific redirected TCR” complex (or “Aspire-TCR”), comprising one or more engineered TCR signaling subunits based on TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, or CD3ζ. Specifically, these engineered TCR signaling subunits may include engineered CD3 zeta (i.e., CD3ζ or CD3z) subunits, and / or engineered target recognition subunits based on any one of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ.

[0124] On the one hand, engineered CD3 zeta subunits are provided, which substantially include co-stimulatory regions (e.g., co-stimulatory domains of CD28, 4-1BB, OX40, etc.) operably linked to or incorporated into intracellular domains of CD3 zeta components (e.g., human CD3z subunits). Expression of engineered CD3 zeta subunits in immune cells (e.g., T cells) can lead to improved properties of immune cells, such as enhanced surface expression of TCR signaling complexes (particularly surface expression of TCRα / TCRβ / TCRγ / TCRδ / CD3ε / CD3γ / CD3δ recognized by specific TCR heterodimers or engineered targets co-expressed therewith), reduced basal / background activation in the absence of antigen stimulation, reduced cytotoxicity against non-target cells, increased activation after antigen stimulation, and / or increased immune cell responses against target cells, etc.

[0125] Engineered CD3z subunits can be modified to further include STAT (e.g., STAT3 or STAT5) binding regions, enabling immune cells expressing such engineered CD3z subunits to have increased proliferative capacity, increased in vivo antitumor efficacy, and / or increased tumor tissue infiltration capacity after repeated antigen stimulation.

[0126] On the other hand, an engineered target recognition subunit is further provided, which substantially includes a target recognition portion (i.e., a target recognition region) operably linked to or operably incorporated into an extracellular domain of a TCR signaling complex subunit (i.e., a TCR subunit), wherein the TCR signaling complex subunit can be any one of TCRα, TCRβ, CD3γ, CD3δ, and CD3ε. According to different embodiments provided in this disclosure, the target recognition portion may comprise a single-chain variable fragment (scFv) or a single-domain antibody (sdAb or nanobody) that specifically recognizes and binds to an epitope of a corresponding antigen expressed or presented on the surface of a specific target cell, or may comprise a ligand portion (i.e., a ligand region) that specifically recognizes and binds to a homologous receptor (i.e., a corresponding ligand receptor) expressed on the surface of a specific target cell. The expression of such engineered target recognition subunits in immune cells (e.g., T cells) enables these engineered immune cells to have specific and effective cytotoxicity against target cells that express these target molecules (i.e., antigens or homologous receptors).

[0127] Specifically, this disclosure provides engineered target-recognizing CD3 epsilon subunits, wherein the target-recognizing portion (e.g., scFv, sdAb, or ligand) is operatively fused to the N-terminus of a CD3 epsilon assembly having a truncated portion (i.e., ΔCD3e) at a region corresponding to its intracellular domain, and further optionally, the C-terminus of the CD3 epsilon assembly (i.e., ΔCD3e) is operatively linked to one or more functional regions (e.g., immune receptor tyrosine-based activation motifs (ITAM), CD28 intracellular domain, CD40 intracellular domain, 4-1BB motif, STAT-binding region, etc.). Depending on different embodiments of the engineered CD3 epsilon subunits (i.e., with different functional regions), they can endow immune cells expressing these subunits with altered or improved properties, such as increased surface expression of the TCR signaling complex, increased amplification capacity after repeated antigen stimulation, altered percentage of memory T cells, increased in vivo antitumor potency, and / or increased tumor infiltration capacity, etc.

[0128] Further details about the aforementioned Aspire-TCR subunit are provided below.

[0129] Engineered CD3 zeta subunit

[0130] In one aspect, this disclosure provides an engineered CD3 zeta (CD3z) subunit that substantially includes a co-stimulatory region operatively linked to or incorporated into a CD3 zeta component. Hereinafter, the CD3 zeta component comprises mammalian CD3 zeta or a functional portion or variant thereof, and the co-stimulatory region is located within an intracellular domain of the engineered CD3 zeta subunit.

[0131] As used herein, the terms “engineered CD3 zeta,” “engineered CD3 zeta subunit,” or “engineered CD3 zeta subunit” refer to a polypeptide based on the mammalian CD3 zeta subunit, or a functional portion or variant thereof, that substantially retains the function of CD3 zeta, for example, the ability to incorporate into the TCR complex and / or mediate TCR intracellular signaling. An example of a mammalian CD3 zeta subunit is the human CD3 zeta subunit, which will be used as the primary embodiment provided below. Thus, according to some embodiments, CD3 zeta comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:1.

[0132] In some embodiments, the engineered CD3z subunit includes a co-stimulatory region (e.g., any co-stimulatory region described herein) operatively linked or fused to a CD3z component (e.g., any CD3z component described herein). In some embodiments, the CD3z component described herein includes human CD3z, a functional portion thereof, or a functional variant thereof. For example, the CD3z component may be full-length human CD3z. In some embodiments, the CD3z component described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:1. In some embodiments, the CD3z component described herein includes an intracellular domain, and the co-stimulatory region described herein is located within the intracellular domain of the CD3z component described herein.

[0133] In some implementations, the engineered CD3 zeta subunit is configured to achieve at least one of the following effects: (1) immune cells expressing the engineered CD3 zeta subunit have reduced activation in the absence of antigen stimulation compared to when immune cells do not express the engineered CD3 zeta subunit; (2) immune cells expressing the engineered CD3 zeta subunit have reduced cytotoxicity against non-target cells compared to when immune cells do not express the engineered CD3 zeta subunit; (3) immune cells expressing the engineered CD3 zeta subunit have increased activation after stimulation compared to when immune cells do not express the engineered CD3 zeta subunit; or (4) immune cells expressing the engineered CD3 zeta subunit have increased immune cell responses against their corresponding target cells compared to when immune cells do not express the engineered CD3 zeta subunit.

[0134] As used herein, the term "co-stimulatory domain" or "co-stimulatory signaling domain" refers to a specific functional portion of an engineered CD3zeta subunit that recruits certain intracellular signaling molecules, thereby conferring at least one of the following capabilities to immune cells: cytotoxicity, stemness (i.e., resistance to exhaustion), memory, persistence, etc. For example, the 4-1BB co-stimulatory domain contains a binding motif for the tumor necrosis factor receptor-associated factor (TRAF) signaling aptamer, thus enabling the recruitment of the TRAF signaling aptamer, resulting in increased T cell memory and persistence. In another example, the CD28 co-stimulatory domain contains a binding motif for certain downstream signaling molecules, thus enabling the recruitment of said downstream signaling molecules, such as phosphatidylinositol-3-kinase (PI3K), growth factor receptor-binding protein 2 (Grb2), and lymphocyte-specific protein tyrosine kinase (Lck), resulting in more effective T cell killing but reduced long-term T cell persistence.

[0135] In this document, the co-stimulatory domain may be derived from natural co-stimulatory immune receptors such as CD28, 4-1BB, OX40, CD2, CD27, CDS, ICAM-1, LFA-1, and ICOS. In some embodiments of the engineered CD3 zeta subunit, the co-stimulatory region comprises the co-stimulatory domain of CD28, or a functional portion or variant thereof; therefore, the first co-stimulatory region may comprise an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:2. In some embodiments of the engineered CD3 zeta subunit, at least one co-stimulatory region comprises a first co-stimulatory region containing a 4-1BB co-stimulatory domain, or a functional portion or variant thereof. Therefore, the first co-stimulatory region may contain an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:3. In other embodiments of the engineered CD3 zeta subunit, at least one co-stimulatory region comprises a first co-stimulatory region containing a co-stimulatory domain, or a functional portion or variant thereof, of any one of receptors OX40, CD2, CD27, CDS, ICAM-1, LFA-1, and ICOS. It should be noted that the co-stimulatory domain may be derived from a non-natural source (i.e., artificially created or synthesized) and may contain engineered binding motifs of certain intracellular signaling molecules, which may be a combination of different binding motifs from different co-stimulatory domains of different immune receptors. Such sequences have been reported in KG Daniels et al., Science 10.1126 / science.abq0225(2022) and WO2022173703A1, each of which is incorporated into this paper in its entirety by reference.

[0136] In some implementations, a functional portion of the costimulatory domain is a part of the costimulatory domain that may contain all or part of the function of the costimulatory domain. For example, the functional portion may contain only one or more binding motifs of certain downstream signaling molecules of a known costimulatory domain. The term "functional variant" refers to a sequence variant of the costimulatory domain, such as those containing sequence substitutions, deletions, insertions, translocations, etc., but retaining all or part of the function of the costimulatory domain.

[0137] In some implementations, in the engineered CD3 zeta subunit provided herein, at least one co-stimulatory region may be located at a different position relative to the CD3 zeta sequence, particularly the intracellular signal transduction domain of CD3 zeta.

[0138] As used herein, the term "intracellular signaling region of CD3 zeta" refers to a portion of the intracellular domain of the CD3 zeta subunit responsible for transducing signals upon stimulation by the TCR complex, and typically includes three immune receptor tyrosine activation motifs (ITAMs). According to some embodiments, one or more of at least one co-stimulatory region is fused at the C-terminus of the intracellular signaling domain of CD3 zeta. However, according to some other embodiments, one or more of at least one co-stimulatory region is located between the transmembrane domain and the intracellular signaling domain of CD3 zeta. However, according to some other embodiments, a first subset of at least one co-stimulatory region is fused at the C-terminus of the intracellular signaling domain of CD3 zeta, and a second subset of at least one co-stimulatory region is located between the transmembrane domain and the intracellular signaling domain of CD3 zeta.

[0139] According to some specific implementations, the engineered CD3 zeta subunit includes a CD28 co-stimulatory region fused to the C-terminus of CD3 zeta. The CD28 co-stimulatory region includes the amino acid sequence shown in SEQ ID NO:2, and CD3 zeta includes the amino acid sequence shown in SEQ ID NO:1.

[0140] According to some specific embodiments, the engineered CD3 zeta subunit includes a 4-1BB intracellular domain fused to the C-terminus of CD3 zeta. The 4-1BB intracellular domain includes the amino acid sequence shown in SEQ ID NO:3, and CD3 zeta includes the amino acid sequence shown in SEQ ID NO:1.

[0141] There is no limitation on the number of at least one co-stimulatory region in the engineered CD3 zeta subunit disclosed herein. For example, according to some embodiments, there is only one co-stimulatory region in the engineered CD3 zeta subunit, while according to some other embodiments, there are more than one co-stimulatory region in the engineered CD3 zeta subunit. In the latter case, the more than one co-stimulatory region may originate from the same immune receptor or from different immune receptors.

[0142] An illustrative example of an engineered CD3 zeta subunit is provided, wherein the engineered subunit contains only one co-stimulatory region (or co-stimulatory domain) within its intracellular domain. Reference Figure 2A The co-stimulatory region is substantially located at the C-terminus of the engineered CD3 zeta subunit; that is, the co-stimulatory region is fused to the C-terminus of the CD3 zeta subunit (SEQ ID NO:1) via a flexible connector (shown as a line connecting the two blocks representing CD3z and the co-stimulatory region). In this document, the flexible connector is optional and may be non-essential in some embodiments.

[0143] The actual location of the co-stimulatory region within the engineered CD3z subunit is not limited, as long as CD3z function transduces TCR signaling after recognition by the engineered TCR complex with a specific antigen / MHC. For example, the co-stimulatory region can be located between the transmembrane domain and the immunoreceptor tyrosine activation domain of the three immunoreceptor tyrosine activation motifs (ITAMs) containing the CD3z subunit.

[0144] In some embodiments, the co-stimulatory region is an intracellular signaling region of a cell surface protein expressed in T cells, or a functional portion or functional variant thereof, which has a co-stimulatory function, i.e., is capable of providing a co-stimulatory signal for the activation, survival, and / or proliferation of T cells. Non-limiting examples of co-stimulatory proteins whose intracellular signaling domains or functional portions thereof (e.g., the functional portion may include one or more signaling motifs) can be used to engineer the CD3 zeta subunit include CD28, 4-1BB, LFA-1, CD4, CCD28, CD27, ICOS, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226.

[0145] In some embodiments, the co-stimulatory region may be derived from CD28 or 4-1BB and may optionally have amino acid sequences having 80-100% sequence identity with SEQ ID NO:2 and 3, respectively. In some embodiments, the co-stimulatory region may optionally be fused to CD3z without any linker, or optionally fused to CD3z via a flexible linker having a length of 1-20 amino acid residues and primarily comprising small-volume amino acid residues such as glycine (“G”) or serine (“S”). According to one specific embodiment, the flexible linker may have a sequence as shown in SEQ ID NO:6 or SEQ ID NO:7.

[0146] In some embodiments, the co-stimulatory region described herein is located between the transmembrane domain and the intracellular domain of the CD3z component described herein. In some embodiments, the co-stimulatory region is a STAT-binding region, such as a STAT3 or STAT5 binding region. In some embodiments, the STAT3 binding region may contain an amino acid sequence having at least 50%, 75%, or 100% sequence identity with YXXQ (SEQ ID NO:22), wherein the two X residues may be the same or different. In some embodiments, the first X residue is R (or any amino acid with similar physical and / or chemical properties, such as H or K), and the second residue is H (or any amino acid with similar physical and / or chemical properties, such as R or K). In some embodiments, the STAT3 binding region may contain an amino acid sequence having at least 50%, 75%, or 100% sequence identity with YRHQ (SEQ ID NO:21). In some embodiments, the STAT5 binding region may contain an amino acid sequence having at least 50%, 75%, or 100% sequence identity with YXXL (SEQ ID NO:23), wherein the two X residues may be the same or different. In some embodiments, the first X residue is L (or any amino acid with similar physical and / or chemical properties, such as A, V, I, M, F, Y, or W), and the second residue is S (or any amino acid with similar physical and / or chemical properties, such as T, N, or Q). In some embodiments, the STAT5 binding region may contain an amino acid sequence having at least 50%, 75%, or 100% sequence identity with YLSL (SEQ ID NO:24). In some embodiments, the STAT-binding region is located in the region of the CD3z component corresponding to positions 150-164 of SEQ ID NO:1, for example, between any two amino acid residues corresponding to positions 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, and 165 of SEQ ID NO:1. In some embodiments, the sequence between the two residues corresponds to a deletion of wild-type human CD3z. In some embodiments, the STAT-binding region is inserted into the CD3z component (e.g., any CD3z component described herein) within its intracellular domain. In some embodiments, the STAT-binding region is inserted into positions 157 and 158 of the CD3z component corresponding to positions SEQ ID NO:1.In some embodiments, the CD3z component, for example, after insertion of a STAT binding region (e.g., any STAT3 or STAT5 binding region described herein), may contain an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:20 (when inserting the STAT3 binding region YRHQ) or SEQ ID NO:29 (when inserting the STAT5 binding region YLSL).

[0147] In some embodiments, one or more co-stimulatory regions are fused to the C-terminus of the CD3 zeta assembly. In some embodiments, one or more co-stimulatory regions are inserted between the transmembrane and intracellular domains of the CD3 zeta assembly. In some embodiments, the engineered CD3 zeta subunit includes one, two, three, or four co-stimulatory regions. In some embodiments, one or more co-stimulatory regions have the same sequence. In some embodiments, one or more co-stimulatory regions are derived from the same co-stimulatory receptor (e.g., 4-1BB). In some embodiments, one or more co-stimulatory regions are derived from different co-stimulatory receptors (e.g., 4-1BB and CD28).

[0148] In some embodiments, the co-stimulatory region contains the amino acid sequence shown in SEQ ID NO:2 (RSKRSRLLHSDYMNMTPRRPGPTRKHYQ) PYAP PRDFAAYRS). It should be noted that the underlined 4-AA portion in SEQ ID NO:2 is a recognized core functional co-stimulatory sequence; however, unexpectedly, we found that adding the C-terminal 28-AA segment rich in positively charged amino acids could improve the phenotype by reducing the base-modal signaling of Aspire-TCR.

[0149] In some embodiments, this document provides an engineered CD3z subunit comprising a human CD3z and CD28 co-stimulatory domain from its N-terminus to its C-terminus. In some embodiments, the engineered CD3z subunit is composed of... Figure 14 The code shown here encodes any construct for designing AH. In some embodiments, the human CD3z described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:1. In some embodiments, the CD28 co-stimulatory domain described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:2.

[0150] In some embodiments, this document provides an engineered CD3z subunit comprising, from its N-terminus to its C-terminus, a modified human CD3z and CD28 co-stimulatory domain. In some embodiments, the engineered CD3z subunit is composed of... Figure 14 The code shown herein encodes any construct for designing IK. In some embodiments, the modified human CD3z comprises a STAT-binding region (e.g., a STAT3 or STAT5 binding region) inserted into its intracellular region (e.g., between positions 157 and 158 of SEQ ID NO:1). In some embodiments, the STAT3 binding region (e.g., SEQ ID NO:22 or SEQ ID NO:21) is used for insertion herein. In some embodiments, the modified human CD3z described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:20. In some embodiments, the CD28 co-stimulatory domain described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:2. In some implementations, the CD3z component described herein consists of or contains the modified human CD3z described herein.

[0151] Engineered target recognition subunit

[0152] In one aspect, this disclosure further provides an engineered target recognition subunit that, when expressed in immune cells, can confer specific targeting of the immune cells against target cells expressing specific target molecules that can be recognized by the engineered target recognition subunit.

[0153] The engineered target recognition subunit contains an extracellular domain that includes a target recognition region. As used herein, the term “target recognition region” may be used interchangeably with “target recognition portion” or “target recognition part” or similar terms, and refers to a portion of the engineered target recognition subunit within its extracellular domain that specifically recognizes and binds to target molecules expressed on target cells (i.e., homologous binding partners), thereby enabling the engineered TCR complex to exert its cytotoxicity against the target cells.

[0154] In some embodiments, the target recognition region may comprise a single-chain variable fragment (scFv) or a single-domain antibody (sdAb or nanobody) that specifically recognizes and binds to an epitope of an antigen (i.e., a target molecule) presented on the surface of a target cell corresponding to the scFv or sdAb. In some embodiments, any scFv or sdAb that can specifically target a target molecule enriched in the target cell can be used as the target recognition region of an engineered TCR complex.

[0155] In some embodiments, the target recognition region may include a ligand, or a functional portion or variant thereof (hereinafter defined as "functional" as capable of binding to a corresponding receptor), which specifically recognizes and binds to a corresponding cell surface receptor expressed on the surface of a target cell. In this case, any ligand, or its receptor-binding portion or functional variant thereof, capable of specifically targeting a target molecule abundant in the target cell can be used as the target recognition region of an engineered target recognition subunit. Hereinafter, the ligand, or its functional portion or variant thereof, may be a natural ligand, but may also be a peptide that has been artificially identified or engineered.

[0156] In some embodiments, the engineered target recognition subunit carrying the aforementioned target recognition region can be fused to the backbone of any one of TRa, TCRb, CD3g, CD3d, or CD3e, or optionally based on an engineered protein that can be incorporated into the TCR complex. In some embodiments, the target recognition region can be located anywhere on the engineered target recognition subunit, as long as it is within its extracellular domain. For example, the target recognition region can be located at the N-terminus of one of the five subunits (i.e., TRa, TCRb, CD3g, CD3d, or CD3e). Figure 2B An embodiment in which the target recognition region (ligand) is fused with CD3e (full-length CD3e or truncated form) via a GS-rich linker (i.e., "GS linker") is shown.

[0157] In some embodiments, the engineered target recognition subunit comprises CD3e. In some embodiments, CD3e comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 5.

[0158] In some embodiments, the target recognition region includes an IL13(E13Y) ligand region (“IL13E13Y”). In some embodiments, the target recognition region includes an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO: 4. In some embodiments, the target recognition region includes anti-ALPP (“F8”) scFv. In some embodiments, the target recognition region includes an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO: 9.

[0159] In some embodiments, the engineered target recognition subunit comprises CD3e fused to the target recognition region via a linker (e.g., a GS-linker). In some embodiments, the linker comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:6 or SEQ ID NO:7.

[0160] In this article, engineered proteins (i.e., cytoskeletal proteins fused to or incorporated into a target recognition region) may include an extracellular domain carrying the target recognition region, a transmembrane domain, and optionally an intracellular domain. Each of these domains may be derived from any of the TCR-CD3 complex subunits, such as TRa, TCRb, CD3g, CD3d, CD3e, and CD3z, or combinations thereof, or may be artificially engineered.

[0161] In some instances of cytoskeletal proteins, the extracellular domains described herein may include the extracellular domain of the CD3e subunit, which is further fused at its N-terminus to the target recognition region in an engineered target recognition subunit; the transmembrane domains described herein may include the transmembrane domain of the CD3e subunit; and the intracellular domains described herein may include the intracellular domain of the CD3z subunit.

[0162] In another instance, the extracellular domain described herein may include the extracellular domain of the CD3g subunit, which is further fused to the target recognition region at its N-terminus; the transmembrane domain described herein may include the transmembrane domain of the CD3e subunit; and the intracellular domain described herein may include the intracellular domain of the CD3z subunit.

[0163] In yet another instance, the extracellular domain described herein may include the extracellular domain of the TCRa subunit, which is further fused to the target recognition region at its N-terminus; the transmembrane domain described herein may include the transmembrane domain of the CD3e subunit; and the intracellular domain described herein may include the intracellular stimulatory domain of the CD3z subunit fused to the intracellular stimulatory domain of the CD3e subunit.

[0164] It should be noted that the engineered proteins described above are merely illustrative examples, and each extracellular domain can be further engineered. For example, intracellular domains may contain 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) intracellular signaling motifs (e.g., ITAMs), each derived from CD3e, CD3g, CD3g, and CD3z, or as engineered ITAMs.

[0165] In some embodiments, the engineered target recognition subunit includes at least one functional region within its intracellular domain. In some embodiments, at least one functional region is fused to the C-terminus of the CD3 epsilon component.

[0166] In some embodiments, at least one functional region comprises an immune receptor tyrosine-based activation motif (ITAM). In some embodiments, the ITAM is derived from CD3z, CD3g, DAP12, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD28, or CD72. In some embodiments, the ITAM comprises: (1) CD3z ITAM3, which comprises at least 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:11; or (2) CD3z ITAM2-3, which comprises at least 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:12. In some embodiments, the ITAM is located at the C-terminus of an engineered CD3 epsilon subunit. In some embodiments, the ITAM is fused to the C-terminus of the CD3 epsilon component, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, the ITAM comprises a STAT binding region, wherein the STAT binding region is a STAT3 binding motif (e.g., any STAT3 binding motif described herein) or a STAT5 binding motif (e.g., any STAT5 binding motif described herein).

[0167] In some embodiments, at least one functional region comprises any one or a combination of the following: (1) an intracellular domain of FcεR1γ containing at least 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:25; (2) an intracellular domain of OX40 containing at least 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:26; (3) an intracellular domain of CD40 containing at least 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:27; (4) an intracellular domain of DAP12 containing at least 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:28; (5) an intracellular domain of 4-1BB containing at least 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:3; and (6) a 4-1BB motif containing at least 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:25. (7) The amino acid sequence of NO:15 is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:17; and (8) The adapter (LAT) motif for activating T cells contains at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0168] In some embodiments, the engineered CD3 epsilon subunit includes a STAT-binding region within its intracellular domain. In some embodiments, the STAT-binding region includes: (1) a STAT3 binding motif containing at least 50%, 75%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:22; or (2) a STAT5 binding motif containing at least 50%, 75%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:23. In some embodiments, the STAT3 binding region described herein may contain an amino acid sequence having at least 50%, 75%, or 100% sequence identity with YXXQ (SEQ ID NO:22), wherein the two X residues may be identical or different. In some embodiments, the first X residue is R (or any amino acid having similar physical and / or chemical properties, such as H or K), and the second residue is H (or any amino acid having similar physical and / or chemical properties, such as R or K). In some embodiments, the STAT5 binding region described herein may comprise an amino acid sequence having at least 50%, 75%, or 100% sequence identity with YXXL (SEQ ID NO:23), wherein the two X residues may be identical or different. In some embodiments, the first X residue is L (or any amino acid having similar physical and / or chemical properties, such as A, V, I, M, F, Y, or W), and the second residue is S (or any amino acid having similar physical and / or chemical properties, such as T, N, or Q). In some embodiments, the STAT binding region comprises a STAT3 binding motif comprising an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the STAT binding region comprises a STAT5 binding motif comprising an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of SEQ ID NO:24.

[0169] In some embodiments, the engineered CD3 epsilon subunit described herein includes an ITAM in its intracellular domain, wherein the ITAM contains a STAT-binding region (e.g., any STAT-binding region described herein). In some embodiments, the ITAM is a CD3z ITAM2-3 motif containing at least 70%, 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:18 (when inserted into a STAT5-binding region YLSL). In some embodiments, the ITAM is a CD3z ITAM3 motif containing at least 70%, 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:19 (when inserted into a STAT5-binding region YLSL). In some embodiments, the ITAM described herein may include an inserted STAT3-binding region (e.g., any STAT3-binding region described herein). In some embodiments, the ITAM described herein may include an inserted STAT5-binding region (e.g., any STAT5-binding region described herein). In some embodiments, the insertion site may be between any two residues within the CD3zITAM2-3 motif or the CD3zITAM3 motif described herein. In some embodiments, the insertion site corresponds to positions 58 and 59 of the CD3zITAM2-3 motif (SEQ ID NO:12). In some embodiments, the insertion site corresponds to positions 27 and 28 of the CD3zITAM3 motif (SEQ ID NO:11). In some embodiments, at least one functional region comprises both an intracellular FcεR1γ domain and an intracellular OX40 domain. In some embodiments, at least one functional region comprises a first complex functional region comprising an intracellular FcεR1γ domain and an intracellular OX40 domain in a direction from the N-terminus to the C-terminus, wherein the first complex functional region comprises at least 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:13. In some embodiments, at least one functional region further comprises ITAM at the C-terminus of the first complex functional region, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, at least one functional region comprises both a CD40 intracellular domain and a DAP12 intracellular domain. In some embodiments, at least one functional region comprises a second complex functional region comprising a CD40 intracellular domain and a DAP12 intracellular domain from the N-terminus to the C-terminus, wherein the second complex functional region comprises an amino acid sequence that is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:14.In some embodiments, at least one functional region further comprises an ITAM at the C-terminus of the second composite functional region, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, the engineered CD3 epsilon subunit comprises a 4-1BB intracellular domain and an ITAM from its N-terminus to its C-terminus, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, at least one functional region comprises both a 4-1BB motif and a LAT motif.

[0170] In some embodiments, the engineered CD3 epsilon subunit includes an ITAM at the C-terminus of the engineered CD3 epsilon subunit. In some embodiments, the ITAM includes CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, the ITAM includes a STAT binding region, wherein the STAT binding region is a STAT3 binding motif or a STAT5 binding motif.

[0171] In some embodiments, at least one functional region includes both the CD40 motif and the LAT motif. In some embodiments, the engineered CD3 epsilon subunit includes ITAM at the C-terminus of the engineered CD3 epsilon subunit. In some embodiments, the ITAM includes CD3z ITAM3 or CD3z ITAM2-3. In some embodiments, the target recognition region is operatively connected to or incorporated into the CD3 epsilon component.

[0172] In some implementations, the target recognition region includes: (1) an antigen-binding region; or (2) a ligand or fragment thereof that binds to a cell surface receptor expressed on a target cell of an immune cell. In some embodiments, the antigen-binding region comprises a single-stranded variable fragment (scFv) that specifically recognizes ALPP, LYPD3, IL13Ra2, BCMA, CD16, CD19, CD20, CD22, CD27, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, mesothelin, NKG2D, phosphatidylinositol proteoglycan-3 (GPC-3), FAP, FRa (folate receptor α), EGFR, EGFR vIII, IL-11Ra (IL11 receptor α), VEGFR-II, B7-H6, and DNAM-1. In some embodiments, the antigen-binding region comprises a single-stranded variable fragment (scFv) targeting ALPP. In some embodiments, the antigen-binding region comprises a single-domain antibody (sdAb or nanobody). In some embodiments, the ligand is selected from the group consisting of: IL13 (E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRIN B2, CTLX, LFA-1, and FSH. In some embodiments, the ligand is IL13 (E13Y).

[0173] In some implementations, this document provides engineered target recognition subunits (e.g., any CD3e subunit described herein).

[0174] In some embodiments, this document provides an engineered CD3e subunit that includes a target recognition region (e.g., any target recognition region described herein) and full-length human CD3e from the N-terminus to the C-terminus. In some embodiments, the engineered CD3e subunit is composed of... Figure 14 The construct shown in the diagram encodes design A. In some embodiments, the target recognition region is scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:5.

[0175] In some embodiments, this document provides an engineered CD3e subunit that includes a target recognition region (e.g., any target recognition region described herein) and a truncated human CD3e from its N-terminus to its C-terminus. In some embodiments, the engineered CD3e subunit is composed of... Figure 14 The diagram shows the encoding of either design B or design I. In some embodiments, the target recognition region is the scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:10.

[0176] In some embodiments, this document provides an engineered CD3e subunit comprising, from its N-terminus to its C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, and CD3z ITAM2-3 (e.g., any CD3z ITAM2-3 motif described herein). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct shown in the diagram encodes design C. In some embodiments, the target recognition region is the scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, CD3zITAM2-3 comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 12.

[0177] In some embodiments, this document provides an engineered CD3e subunit comprising, from the N-terminus to the C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, an FcεR1γ intracellular domain (e.g., any FcεR1γ intracellular domain described herein), an OX40 intracellular domain (e.g., any OX40 intracellular domain described herein), and CD3z ITAM3 (e.g., any CD3z ITAM3 motif described herein). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct shown in the diagram encodes design D. In some embodiments, the target recognition region is the scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:10. In some embodiments, the FcεR1γ intracellular domain and the OX40 intracellular domain together comprise an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:13. In some embodiments, CD3z ITAM3 comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:11.

[0178] In some embodiments, this document provides an engineered CD3e subunit comprising, from its N-terminus to its C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, a CD40 intracellular domain (e.g., any CD40 intracellular domain described herein), a DAP12 intracellular domain (e.g., any DAP12 intracellular domain described herein), and CD3z ITAM3 (e.g., any CD3z ITAM3 motif described herein). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct shown in the diagram encodes design E. In some embodiments, the target recognition region is scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the CD40 intracellular domain and the DAP12 intracellular domain together comprise an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 14. In some embodiments, CD3z ITAM3 comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 11.

[0179] In some embodiments, this document provides an engineered CD3e subunit comprising, from the N-terminus to the C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, a 4-1BB intracellular domain (e.g., any 4-1BB intracellular domain described herein), and CD3z ITAM3 (e.g., any CD3z ITAM3 motif described herein). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct shown in the diagram encodes the design F. In some embodiments, the target recognition region is scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:10. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:3. In some embodiments, CD3z ITAM3 comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:11.

[0180] In some embodiments, this document provides an engineered CD3e subunit comprising, from its N-terminus to its C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, a 4-1BB motif (e.g., any 4-1BB motif described herein), a LAT motif (e.g., any LAT motif described herein), and CD3z ITAM3 (e.g., any CD3z ITAM3 motif described herein). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct encoding design G is shown in the diagram. In some embodiments, the target recognition region is scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:10. In some embodiments, the 4-1BB motif comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:15. In some embodiments, the LAT motif comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:16. In some embodiments, CD3z ITAM3 contains an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 11.

[0181] In some embodiments, this document provides an engineered CD3e subunit comprising, from its N-terminus to its C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, a CD40 motif (e.g., any CD40 motif described herein), a LAT motif (e.g., any LAT motif described herein), and CD3z ITAM3 (e.g., any CD3z ITAM3 motif described herein). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct encoding design H is shown in the diagram. In some embodiments, the target recognition region is the scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:10. In some embodiments, the CD40 motif comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:17. In some embodiments, the LAT motif comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:16. In some embodiments, CD3z ITAM3 contains an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 11.

[0182] In some embodiments, this document provides an engineered CD3e subunit comprising, from its N-terminus to its C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, and CD3z ITAM2-3 (e.g., any CD3z ITAM2-3 motif described herein having a STAT5 binding region). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct shown in the diagram encodes design J. In some embodiments, the target recognition region is the scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, CD3z ITAM2-3 (with a STAT5 binding region) comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 18.

[0183] In some embodiments, this document provides an engineered CD3e subunit comprising, from its N-terminus to its C-terminus, a target recognition region (e.g., any target recognition region described herein), a truncated human CD3e, a 4-1BB motif (e.g., any 4-1BB motif described herein), a LAT motif (e.g., any LAT motif described herein), and CD3z ITAM3 (e.g., any CD3z ITAM3 motif described herein with a STAT5 binding region). In some embodiments, the engineered CD3e subunit is composed of... Figure 14The construct encoding design K is shown in the diagram. In some embodiments, the target recognition region is the scFv. In some embodiments, the scFc described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:30. In some embodiments, the truncated human CD3e described herein does not contain the intracellular domain of human CD3e (e.g., the functional intracellular domain of human CD3e). In some embodiments, the truncation (e.g., any modification, deletion, insertion, and / or substitution) is in the region corresponding to positions 151 to 179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e does not contain the amino acid sequence corresponding to positions 151-179 of SEQ ID NO:5. In some embodiments, the truncated human CD3e described herein comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:10. In some embodiments, the 4-1BB motif comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:15. In some embodiments, the LAT motif comprises an amino acid sequence having at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity with SEQ ID NO:16. In some embodiments, CD3z ITAM3 (with a STAT5 binding region) contains an amino acid sequence that has at least 70%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 19.

[0184] Engineered TCR complex system

[0185] In some embodiments, this document provides an engineered TCR complex system comprising an engineered CD3e subunit (e.g., any CD3e subunit described herein). In some embodiments, the engineered TCR complex system further comprises an engineered CD3z subunit (e.g., any CD3z subunit described herein).

[0186] In some embodiments, the engineered CD3z subunit described herein includes a co-stimulatory region (e.g., any co-stimulatory region described herein). In some embodiments, the co-stimulatory region described herein is human CD28, its functional portion, or a functional variant. In some embodiments, the co-stimulatory region described herein is human 4-1BB, its functional portion, or a functional variant.

[0187] In some embodiments, one or both of the engineered CD3e subunit and engineered CD3z subunit described herein contain a STAT-binding region (e.g., any STAT-binding region described herein). In some embodiments, the STAT-binding region described herein is within an intracellular domain of the engineered CD3e subunit and engineered CD3z subunit described herein. Specifically, in some embodiments, the engineered CD3e subunit described herein contains a STAT5-binding region, and the engineered CD3z subunit described herein contains a STAT3-binding region; in some embodiments, the engineered CD3e subunit described herein contains a STAT3-binding region, and the engineered CD3z subunit described herein contains a STAT5-binding region. In some embodiments, the engineered CD3e subunit described herein contains a STAT5-binding region or a STAT3-binding region, and the engineered CD3z subunit described herein does not contain a STAT5-binding region or a STAT3-binding region. In some embodiments, the engineered CD3z subunit described herein contains a STAT5-binding region or a STAT3-binding region, and the engineered CD3e subunit described herein does not contain a STAT5-binding region or a STAT3-binding region.

[0188] In some embodiments, when the engineered CD3e subunit described herein is expressed in a T cell population, it satisfies at least one of the following conditions compared to when the STAT-binding region is not present in either the engineered CD3 epsilon subunit or the engineered CD3 zeta subunit: the T cell population contains a higher percentage (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold) of terminally differentiated effector T cells; the T cell population exhibits an increased percentage (e.g., at least 10%, 20%, 30%, or 40%) of terminally differentiated effector T cells in vitro. Antigen-specific stimulation of 50%, 60%, 70%, 80%, 90%, 1, 2, 5, 10, 20, 50, or 100 times; and T cell populations showing increased expansion in vitro after repeated stimulation with antigens (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 5, 10, 20, 50, or 100 times).

[0189] In some embodiments, the engineered CD3e and engineered CD3z subunits described herein can interact with endogenous (e.g., unmodified) TCR subunits, endogenous (e.g., unmodified) CD3g subunits, and / or endogenous (e.g., unmodified) CD3d subunits to form a functional TCR-CD3 complex.

[0190] Regulating the activity of immune cells

[0191] In one aspect, this disclosure provides a method for regulating the activity of immune cells.

[0192] In some embodiments, the method substantially includes the step of expressing an exogenous CD3 zeta subunit in immune cells. In this document, the exogenous CD3 zeta subunit may be simply the mammalian CD3 zeta subunit, or a functional portion or variant thereof, or more preferably an engineered CD3 zeta subunit according to any embodiment of the engineered CD3 zeta subunit as described above.

[0193] In some embodiments, the method is configured such that, after the above steps are applied to immune cells, at least one of the following effects is achieved:

[0194] (1) Immune cells expressing engineered CD3 zeta subunits showed reduced activation in the absence of TCR complex antigen stimulation compared to when immune cells did not express engineered CD3 zeta subunits.

[0195] (2) Immune cells expressing engineered CD3 zeta subunits exhibited reduced cytotoxicity against non-target cells compared to immune cells that did not express engineered CD3 zeta subunits.

[0196] (3) Immune cells expressing engineered CD3 zeta subunits showed increased activation upon stimulation by the TCR complex compared to immune cells that did not express the engineered CD3 zeta subunit; or

[0197] (4) Immune cells expressing engineered CD3 zeta subunits have increased immune cell responses against their corresponding target cells compared to immune cells that do not express engineered CD3 zeta subunits.

[0198] In some implementations, the immune cells may be T lymphocytes (i.e., T cells), tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, or NK T cells.

[0199] According to some implementation schemes of the method, immune cells express engineered TCRs or CARs.

[0200] engineered cells

[0201] This disclosure provides engineered cells (e.g., T cells) comprising the engineered CD3 zeta subunit, engineered target recognition subunit, or engineered TCR complex described herein. These engineered cells can be used to treat a variety of conditions or diseases as described herein (e.g., viral infections, cancer, virus-induced conditions).

[0202] In various embodiments, the engineered cells may be obtained from, for example, humans and non-human animals. In various embodiments, the engineered cells may be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. Preferably, the cells are derived from humans, rats, or mice. More preferably, the cells are derived from humans. In various embodiments, the engineered cells are blood cells. Preferably, the cells are white blood cells (e.g., T cells), lymphocytes, or any other suitable blood cell type. In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are T cells, B cells, or NK cells.

[0203] In some embodiments, the cells are T cells. In some embodiments, the T cells may express a cell surface receptor that recognizes a specific antigenic portion on the surface of the target cell. The cell surface receptor may be a wild-type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing an antigenic portion associated with the target cell. T cells may be obtained by various methods known in the art, such as in vitro culture of T cells isolated from a patient (e.g., tumor-infiltrating lymphocytes). TCR-modified T cells may be obtained by transducing T cells with a viral vector (e.g., isolated from peripheral blood of a patient). In some embodiments, the T cells are TCR-modified T cells. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are type 1 helper T cells and type 2 helper T cells. In some embodiments, the T cells expressing the receptor are αβ-T cells. In alternative embodiments, the T cells expressing the receptor are γδ-T cells.

[0204] In some embodiments, the cells are NK cells. In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used to introduce binding molecules such as TCRs may be isolated from a sample, such as a biological sample, for example, obtained from or derived from a subject. In some embodiments, the subject from whom cells are isolated is a subject with a disease or condition or who requires or will be given cell therapy. In some embodiments, the subject is a person who requires a specific therapeutic intervention such as adoptive cell therapy (in which cells are isolated, processed, and / or engineered).

[0205] In some embodiments, the cells are stem cells, such as multipotent stem cells and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells can be primary cells, such as cells isolated directly from the subject and / or cells isolated from the subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain the desired cell type (e.g., T cells).

[0206] Different cell types can be obtained from appropriate isolation methods. Isolation methods include separating different cell types based on the expression or presence of one or more specific molecules (such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids) in the cells. In some embodiments, any known isolation method based on such markers can be used. In some embodiments, isolation is based on affinity or immunoaffinity. For example, in some aspects, isolation includes separating cells and cell populations based on the cellular expression or expression level of one or more markers (typically cell surface markers), for example, by incubating with an antibody or binding partner that specifically binds to such a marker, followed typically by a washing step, and separating cells that have bound the antibody or binding partner from those that have not.

[0207] Such separation steps can be based on positive and / or negative selection, in which positive selection retains cells that have bound to the reagent for further use, and in negative selection retains cells that have not bound to the antibody or bound coupler. In some instances, both fractions are retained for further use. In some respects, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, allowing for optimal separation based on markers expressed by cells other than the desired population.

[0208] Methods, nucleic acids, compositions, and kits for expressing binding molecules and for generating genetically engineered cells expressing such binding molecules are also provided. Genetic engineering typically involves introducing nucleic acids encoding therapeutic molecules such as TCRs, CARs (e.g., TCR-like CARs), peptides, or fusion proteins into cells, such as through retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is accomplished by the following steps: first, stimulating cells, such as by combining cells with stimuli that induce responses such as proliferation, survival, and / or activation, for example, as measured by the expression of cytokines or activation markers; subsequently, transducing the activated cells; and expanding them in culture to a number sufficient for clinical application.

[0209] In some embodiments, recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV), are used to transfer recombinant nucleic acids into cells. In some embodiments, recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors, are used to transfer recombinant nucleic acids into T cells. In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, for example, a retroviral vector derived from Moloney's murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are generally amphotropic, meaning they can infect host cells of several species, including humans. In some embodiments, the vector is a lentiviral vector. In some embodiments, the recombinant nucleic acid is transferred into T cells via electroporation. In some embodiments, the recombinant nucleic acid is transferred into T cells via translocation. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-assisted microparticle bombardment, and strontium phosphate DNA coprecipitation. Many of these methods are described, for example, in WO2019195486, which is incorporated herein by reference in its entirety.

[0210] In some embodiments, when engineered TCR complexes are engineered into human T cells, they may compete with and / or form mispairings with endogenous TCR complex components (e.g., TCR or CD3), which may in some respects reduce the signaling, activity, and / or expression of the engineered TCR complex, ultimately leading to reduced activity of the engineered cells. The engineered cells may be genetically modified. In some embodiments, the engineered cells may contain genetically disrupted genes encoding endogenous TCR complex components. In some embodiments, the engineered cells do not express endogenous TCR complex components.

[0211] Also provided are engineered cell populations, compositions containing such cells and / or rich in such cells, such as wherein cells expressing the binding molecule comprise at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher percentages of the total cells in the composition or of a specific type of cells such as T cells, CD8+, or CD4+ cells.

[0212] Recombinant vector

[0213] This disclosure also provides recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), wherein a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains polynucleotides and / or a vector containing polynucleotides), and recombinant polypeptides or fragments thereof are generated by recombinant techniques.

[0214] As used herein, “vector” refers to any construct capable of delivering one or more polynucleotides of interest to a host cell when introduced into the host cell. An “expression vector” is capable of delivering one or more polynucleotides of interest and expressing them as encoded polypeptides in a host cell that has already been introduced with an expression vector. Therefore, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operatively linked to regulatory elements such as promoters, enhancers, and / or poly-A tails at or near the integration site of the polynucleotide of interest within the vector or in the host cell genome, such that the polynucleotide of interest will be translated in the host cell in which the expression vector has been introduced.

[0215] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0216] This disclosure provides a recombinant vector containing nucleic acid constructs suitable for genetic modification of cells, which can be used to treat pathological diseases or conditions.

[0217] Any vector or vector type can be used to deliver genetic material into cells. These vectors include, but are not limited to, plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). Viral vectors may include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenovirus vectors, foam virus vectors, recombinant adeno-associated virus (AAV) vectors, hybridization vectors, and plasmid transposons (e.g., the Sleeping Beauty transposon system and the PiggyBac transposon system) or integrase-based vector systems. Other vectors known in the art may also be used in conjunction with the methods described herein.

[0218] In some embodiments, the vector is a viral vector. The viral vector can be grown in a culture medium specifically designed for the manufacture of viral vectors. According to the embodiments described herein, any suitable growth medium and / or supplements for growing viral vectors can be used.

[0219] In some implementations, the vector used is a recombinant retroviral vector. Retroviral vectors are capable of directing the expression of nucleic acid molecules of interest. Retroviruses exist in their viral capsid as RNA and form a double-stranded DNA intermediate as they replicate in a host cell. Similarly, retroviral vectors exist in both RNA and double-stranded DNA forms. Retroviral vectors also include DNA forms containing recombinant DNA fragments and RNA forms containing recombinant RNA fragments. The vector may contain at least one transcription promoter / enhancer, or other elements controlling gene expression. Such vectors may also contain packaging signals, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites suitable for the retrovirus used. Long terminal repeats (LTRs) are identical DNA sequences repeated many times (e.g., hundreds or thousands of times) at either end of a retrotransposon or proviral DNA formed by reverse transcription of retroviral RNA. Viruses use them to insert their genetic material into the host genome. Optionally, the vector may also contain a signal guiding polyadenylation, selective markers such as ampicillin resistance, neomycin resistance, TK, hygromycin resistance, fosetyl-aluminum resistance, histamine resistance, or DHFR, and one or more restriction sites and translation termination sequences. For example, such vectors may contain a 5' LTR, a leader sequence, an tRNA binding site, a packaging signaling pathway, a start point for second-strand DNA synthesis, and a 3' LTR or a portion thereof. Additionally, the retroviral vectors used herein may also refer to recombinant vectors generated by removing the retroviral gag, pol, and env genes and replacing them with the gene of interest.

[0220] In some embodiments, the vector may contain additional nucleic acid encoding a repressor protein (e.g., a checkpoint inhibitor). In various embodiments, the cell expresses a genetically engineered antigen receptor and a repressor protein. In various embodiments, the repressor protein is constitutively expressed.

[0221] In some embodiments, the vector or construct may contain a single promoter driving the expression of one or more nucleic acid molecules. In some embodiments, such a promoter may be polycistronic (bicistronic or tricistronic). For example, in some embodiments, the transcription unit may be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of the gene product via information from a single promoter. Alternatively, in some cases, a single promoter may guide the expression of RNA containing two or three genes separated from each other in a single open reading frame (ORF) by sequences encoding self-cleaving peptides (e.g., P2A or T2A) or protease recognition sites (e.g., furin protease). Thus, the ORF encodes a single polyprotein that cleaves into individual proteins during translation (in the case of 2A, such as T2A) or posttranslational. In some cases, peptides such as T2A may cause ribosome skipping (ribosome jumping) of the synthesis of a peptide bond at the C-terminus of a 2A element, resulting in separation between the end of the 2A sequence and the next downstream peptide.

[0222] Various cell lines can be used in conjunction with the vectors described herein. Exemplary eukaryotic cells that can be used to express peptides include, but are not limited to: COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells; Cells; and NSO cells. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications of the binding molecules. For example, in some embodiments, CHO cells produce peptides with higher levels of sialylated peptides than the same peptides produced in 293 cells.

[0223] In one aspect, this disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising:

[0224] (1) The engineered CD3 zeta subunit described in this paper; and / or

[0225] (2) The engineered target recognition subunit described in this paper.

[0226] In some embodiments, the peptide comprises the engineered CD3 zeta subunit and the engineered target recognition subunit described herein. In some embodiments, the engineered CD3 zeta subunit and the engineered target recognition subunit described herein are linked via a T2A linker.

[0227] In one aspect, this disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising:

[0228] (1) The engineered CD3 zeta subunit described in this paper; and / or

[0229] (2) The engineered target recognition subunit described in this paper.

[0230] In some embodiments, the peptide comprises the engineered CD3 zeta subunit and the engineered target recognition subunit described herein. In some embodiments, the engineered CD3 zeta subunit and the engineered target recognition subunit described herein are linked via a T2A linker.

[0231] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv and CD3e; and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domains.

[0232] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv and truncated CD3e; and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domains.

[0233] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, and CD3z ITAM (e.g., CD3z ITAM2-3); and the engineered CD3zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domains.

[0234] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, FceR1g intracellular domain, OX40 intracellular domain, and CD3z ITAM (e.g., CD3z ITAM3); and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domains.

[0235] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, OX40 intracellular domain, DAP12 intracellular domain, and CD3z ITAM (e.g., CD3z ITAM3); and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domains.

[0236] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, 4-1BB intracellular domain, and CD3z ITAM (e.g., CD3zITAM3); and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domain.

[0237] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, 4-1BB motif, LAT motif, and CD3z ITAM (e.g., CD3zITAM3); and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domains.

[0238] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, CD40 intracellular domain, LAT motif, and CD3z ITAM (e.g., CD3zITAM3); and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z and CD28 co-stimulatory domain.

[0239] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv and truncated CD3e; and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z component (e.g., SEQ ID NO:20) and CD28 co-stimulatory domain.

[0240] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, and CD3z ITAM (e.g., CD3z ITAM2-3); and the engineered CD3zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z component (e.g., SEQ ID NO:20) and CD28 co-stimulatory domain.

[0241] In some implementations, the engineered target recognition subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: scFv, truncated CD3e, 4-1BB motif, LAT motif, and CD3z ITAM (e.g., CD3zITAM3-LHYLSLMQ); and the engineered CD3 zeta subunit (e.g., from the N-terminus to the C-terminus) comprises one or more of the following: CD3z component (e.g., SEQ ID NO:20) and CD28 co-stimulatory domain.

[0242] In one aspect, this disclosure relates to a vector comprising one or more nucleic acids as described herein. In another aspect, this disclosure also relates to a vector comprising two nucleic acids as described herein.

[0243] As used herein, the terms “linker” (L), “linker domain”, or “linker region” refer to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links any domain / region together. Linkers may consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not spatially interfere with each other. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A, T2A), 2A-like linkers, or their functional equivalents, and combinations thereof. In some embodiments, linkers include 2A-like linkers for pyroviruses, the CHYSEL sequence of porcine cyclovir (P2A), the Thysaasigna virus (T2A), or combinations thereof, variants, and functional equivalents. Other linkers will be apparent to those skilled in the art and may be used in the methods described herein.

[0244] This disclosure also provides nucleic acid sequences comprising nucleotide sequences encoding any engineered TCR complex, engineered CD3 zeta subunit, or engineered target recognition subunit (including, for example, its functional portions and functional variants, peptides, or proteins as described herein).

[0245] As used herein, “nucleic acid” may include “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from a natural source, and may contain natural, non-natural, or modified nucleotides. Furthermore, nucleic acids contain complementary DNA (cDNA). Generally, it is preferred that nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, in certain circumstances, it may be appropriate for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions.

[0246] Nucleic acids as described herein can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides. In some of these embodiments, the nucleotide sequence is codon-optimized.

[0247] This disclosure also provides nucleic acids comprising nucleotide sequences complementary to the nucleotide sequences of any nucleic acid described herein, or nucleotide sequences that hybridize under stringent conditions to the nucleotide sequences of any nucleic acid described herein.

[0248] In some embodiments, the nucleotide sequences encoding the engineered CD3 zeta subunit and the nucleotide sequences encoding the engineered target recognition subunit are separated by a peptide sequence that induces ribosome jumping. In some embodiments, the peptide that induces ribosome jumping is a P2A or T2A peptide. In some embodiments, the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.

[0249] In some embodiments, the engineered CD3 zeta subunit, engineered target recognition subunit, or engineered TCR complex described herein is encoded by a codon-optimized nucleotide sequence. In some embodiments, the engineered target recognition subunit or engineered TCR complex further comprises a signal peptide. In particular embodiments, the engineered CD3 zeta subunit, engineered target recognition subunit, or engineered TCR complex described herein is isolated, purified, or recombinant. In some such embodiments, the engineered CD3 zeta subunit, engineered target recognition subunit, or engineered TCR complex described herein is recombinant. In some such embodiments, the engineered CD3 zeta subunit, engineered target recognition subunit, or engineered TCR complex described herein is human.

[0250] This disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleotide sequence described herein. The list includes amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any amino acid sequence as described herein. In some embodiments, this disclosure relates to nucleotide sequences encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence as described herein.

[0251] In some embodiments, the nucleic acid sequence comprises at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence comprises at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0252] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies introduced for optimal alignment and the length of each vacancy.

[0253] Methods for preparing engineered cells

[0254] This disclosure provides methods or processes for manufacturing and using engineered cells to treat pathological diseases or conditions.

[0255] The cells used to introduce the engineered TCR complex can be isolated from a sample, such as a biological sample, for example, obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated is a subject with a disease or condition or who requires or will be given cell therapy. In some embodiments, the subject is a person who requires a specific therapeutic intervention such as adoptive cell therapy (in which cells are isolated, processed, and / or engineered).

[0256] Therefore, in some embodiments, the cells are primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from the subject, as well as samples produced by one or more processing steps such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, and tissue and organ samples, including processed samples derived therefrom.

[0257] In some respects, the samples from which cells are derived or isolated are blood or blood-derived samples, or are derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestine, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. In the context of cell therapy, such as adoptive cell therapy, samples include those from autologous and allogeneic sources.

[0258] In some embodiments, the cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from xenogeneic sources, such as mice, rats, or non-human primates.

[0259] In some embodiments, blood cells collected from the subject are washed, for example, to remove plasma fractions and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is performed by a semi-automatic "flow-through" centrifuge. In some aspects, the washing step is performed by tangential flow filtration (TFF). In some embodiments, after washing, the cells are resuspended in various biocompatible buffers, such as, for example, calcium-free buffers. 2+ / Mg 2+ PBS. In some embodiments, components of the blood cell sample are removed, and the cells are resuspended directly in the culture medium. In some embodiments, the method includes density-based cell separation methods, such as preparing leukocytes from peripheral blood by lysing red blood cells and centrifuging via Percoll or Ficoll gradient.

[0260] In some embodiments, the method includes one or more of the following steps: for example, isolating T cells from a patient's blood; transducing a population of T cells with a viral vector comprising a nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and / or infusing the expanded cells into a patient, wherein the engineered cells (e.g., engineered T cells) will seek out and destroy antigen-positive tumor cells. In some embodiments, the nucleic acid construct further comprises a sequence encoding a repressor protein. In some embodiments, these engineered cells (e.g., engineered T cells) can block PD-1 / PD-L1 immunosuppression and enhance anti-tumor immune responses. In some embodiments, the method further includes: transfecting T cells with a viral vector containing the nucleic acid construct.

[0261] In some embodiments, the method involves introducing any of the vectors described herein into cells, either in vitro or ex vivo. In some embodiments, the vector is a viral vector, and the introduction is performed via transduction. In some embodiments, the method further involves introducing one or more reagents into cells, each of which is independently capable of inducing genetic disruption of a gene encoding a component of the endogenous TCR complex. In some embodiments, one or more reagents are repressive nucleic acids (e.g., siRNA). In some embodiments, one or more reagents are fusion proteins comprising a DNA-targeting protein and a nuclease or RNA-directed nuclease (e.g., clustered, regularly spaced short palindromic nucleic acid (CRISPR)-associated nuclease).

[0262] T cell transfection can be achieved by any standard method, such as calcium phosphate, electroporation, liposome-mediated transfer, microinjection, biological projectile particle delivery systems, or any other method known to the technician. In some implementations, T cell transfection is performed using the calcium phosphate method.

[0263] According to the various embodiments described herein, this disclosure provides immunotherapies for tumors. In some embodiments, engineered cells (e.g., engineered T cells) recognize tumor-associated antigens. In some embodiments, these engineered cells (e.g., engineered T cells) exhibit stronger anti-tumor responses and reduced T cell exhaustion.

[0264] This disclosure provides methods for creating personalized anti-tumor immunotherapies. In some embodiments, engineered cells can be generated from a patient's blood cells. These engineered cells are then infused into the patient as a cell therapy product. This product can be used in any patient with a tumor.

[0265] Treatment

[0266] The methods disclosed herein can be used for various therapeutic purposes. In one aspect, this disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment may halt, slow, retard, or inhibit cancer progression. In some embodiments, the treatment may result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0267] In one aspect, this disclosure is characterized by a method of administering therapeutically effective amounts of engineered cells expressing an engineered TCR complex, an engineered CD3 zeta subunit, or an engineered target recognition subunit to a subject in need (e.g., a subject who has, is identified, or is diagnosed with, cancer).

[0268] In some implementations, the subject has a solid tumor. In some implementations, the subject has breast cancer (e.g., triple-negative breast cancer), carcinoid tumor, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematologic malignancy. In some implementations, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer.

[0269] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0270] Furthermore, this disclosure provides methods for treating an infection or infection-related condition in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit disease progression. These methods typically involve administering a therapeutically effective amount of the genetically engineered cells disclosed herein to a subject in need. In some embodiments, the disease or condition being treated is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoan infections, immunodeficiency, human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, and BK polyomavirus.

[0271] As used herein, "effective amount" or "therapeutic effective amount" means an amount or dose sufficient to achieve a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease (e.g., cancer). Effective amounts will vary depending on factors such as the age and weight of the subject to be administered the therapeutic agent and / or therapeutic composition, the severity of symptoms, and the route of administration, and therefore can be determined on an individual basis.

[0272] An effective amount may be administered in one or more applications. For example, an effective amount of the composition is an amount sufficient to reduce, terminate, stabilize, reverse, inhibit, slow, and / or delay cancer progression in a patient, or an amount sufficient to reduce, terminate, stabilize, reverse, slow, and / or delay the in vitro proliferation of cells (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, an effective amount can vary depending on, in particular, the patient's medical history and other factors such as the type (and / or dosage) of the composition used.

[0273] The effective dosage and schedule of administration can be determined empirically, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dosage that must be administered will vary, for example, depending on the mammal to be treated, the route of administration, the specific type of therapeutic agent, and other drugs administered to the mammal. Guidance on selecting an appropriate dosage can be found in the literature. Furthermore, treatment does not necessarily result in 100% or complete cure or prevention of disease or condition. A variety of treatment / prevention methods with varying degrees of therapeutic effect are available, which are considered potentially advantageous by those skilled in the art.

[0274] In any of the methods described herein, engineered cells and / or at least one additional therapeutic agent may be administered to a subject at least once weekly (e.g., once weekly, twice weekly, three times weekly, four times weekly, once daily, twice daily, or three times daily). In some embodiments, at least two different engineered cells (e.g., cells expressing different binding molecules) are administered in the same composition (e.g., a liquid composition). In some embodiments, engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, engineered cells and at least one additional therapeutic agent are administered in two different compositions. In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0275] In some implementations, one or more additional therapeutic agents may be administered to the subject before, simultaneously with, or after the administration of engineered cells.

[0276] In some implementations, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may be a checkpoint inhibitor (CPI). In some implementations, the checkpoint inhibitor is an inhibitory protein, such as an antibody or its antigen-binding fragment. Checkpoint inhibitors can inhibit or block one or more immune checkpoints, including, for example, PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, prolactin, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, and combinations thereof. In some embodiments, the inhibitory protein blocks PD-1 or PD-L1. In various embodiments, the inhibitory protein comprises anti-PD-1scFv. The inhibitory protein is capable of causing a decrease in the expression of PD-1 or PD-L1, and / or inhibiting the upregulation of PD-1 or PD-L1 in T cells in a population, and / or physically inhibiting the formation of the PD-1 / PD-L1 complex and subsequent signal transduction. In some embodiments, the inhibitory protein blocks PD-1. In some embodiments, additional therapeutic agents are anti-OX40 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-LAG-3 antibodies, anti-TIGIT antibodies, anti-BTLA antibodies, anti-CTLA-4 antibodies, or anti-GITR antibodies. In some implementations, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-CD20 antibody (e.g., rituximab), an anti-EGFR antibody (e.g., cetuximab), an anti-CD319 antibody (e.g., elotuzumab), or an anti-PD1 antibody (e.g., nivolumab).

[0277] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat). In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of activated hedgehog signaling pathways, and agents that selectively degrade estrogen receptors.

[0278] In some implementations, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0279] In some implementations, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0280] In some implementations, carboplatin, albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject. In some implementations, an additional therapeutic agent is selected from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, vinblastine, and / or combinations thereof.

[0281] Compositions and Formulations

[0282] This disclosure provides compositions (including pharmaceutical compositions and therapeutic compositions) containing engineered cells produced by the methods disclosed herein. Methods for administering engineered cells and compositions thereof to a subject (e.g., a patient), such as therapeutic methods, are also provided.

[0283] Compositions comprising engineered cells for administration are provided, including pharmaceutical compositions and formulations, such as unit dosage form compositions comprising a number of cells for administration at a given dose or in portions thereof. The pharmaceutical compositions and formulations may contain one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition contains at least one additional therapeutic agent.

[0284] A pharmaceutically acceptable carrier is a component in a drug composition other than the active ingredient. A pharmaceutically acceptable carrier does not interfere with the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Drug formulation refers to the process of combining different substances and / or reagents to produce a final drug product. Formulation studies involve developing preparations of a drug that are acceptable to patients. Furthermore, the form of the preparation ensures that the biological activity of the active ingredient contained therein is effective and does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0285] In some embodiments, the choice of carrier is determined in part by the specific cells (e.g., T cells or NK cells) and / or by the method of administration. A variety of suitable formulations are available. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount from about 0.0001% by weight to about 2% by weight of the total composition. For example, carriers are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (Residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0286] Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used. Buffers or mixtures thereof are typically present in amounts from about 0.001% by weight to about 4% by weight of the total composition. Methods for preparing administerable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st edition (May 1, 2005).

[0287] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for a specific indication, disease, or condition treated with engineered cells, preferably those active ingredients having activities complementary to engineered cells, wherein the individual activities do not adversely affect each other. Such active ingredients are suitably present in a combination of amounts effective for the intended purpose. Therefore, in some embodiments, the pharmaceutical composition may further comprise other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, and / or vinblastine.

[0288] In some embodiments, the pharmaceutical composition contains an amount of engineered cells effective in treating or preventing a disease or condition (such as a therapeutically effective amount or a preventatively effective amount). In some embodiments, the therapeutic or preventative efficacy is monitored by periodically evaluating the treated subject. The desired dose can be delivered by a single bolus injection of engineered cells, by multiple bolus injections of engineered cells, or by continuous infusion of engineered cells.

[0289] Engineered cells and compositions can be administered using standard application techniques, formulations, and / or devices. The administration of engineered cells can be autologous or allogeneic. For example, immunoreactive T cells or progenitor cells can be obtained from a subject and, after being genetically modified according to the various embodiments described herein, administered to the same subject or different compatible subjects. Peripheral blood-derived immunoreactive T cells or their progeny (e.g., in vivo, ex vivo, or in vitro) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. Typically, when administering a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoreactive cells), it is usually formulated in a unit-dose injectable form (solution, suspension, emulsion).

[0290] The formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parentereal" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, engineered cells are administered to a subject via peripheral systemic delivery, such as intravenous, intraperitoneal, or subcutaneous injection.

[0291] In some embodiments, the composition is provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH in some respects. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are slightly easier to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium, containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0292] Sterile injection solutions can be prepared by incorporating engineered cells into a solvent, such as by mixing with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, or dextrose. Depending on the route of administration and the desired product, the composition may contain excipients such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling agents or viscosity-enhancing additives, preservatives, flavoring agents, and / or coloring agents. In some respects, suitable products can be prepared with reference to standard literature.

[0293] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Antimicrobial activity can be ensured through various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Extended absorption of injectable drug forms can be achieved by using delayed-absorption agents such as aluminum monostearate and gelatin.

[0294] Formulations intended for internal administration are typically sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

[0295] The compositions or pharmaceutical compositions described herein may be included in containers, packages, or dispensers together with the instructions for use.

[0296] Application method

[0297] Methods for administering engineered cells, populations, and compositions are also provided, as well as uses of such cells, populations, and compositions for treating or preventing diseases, conditions, and ailments, including cancer. In some embodiments, the methods described herein may reduce the risk of developing the diseases, conditions, and ailments described herein.

[0298] In some embodiments, the engineered cells, populations, and compositions described herein are administered to a subject or patient with a specific disease or condition, to be treated, for example, via adoptive cell therapy such as adoptive T-cell therapy. In some embodiments, cells and compositions prepared by the provided methods, such as engineered compositions after incubation and / or other processing steps and end-of-production compositions, are administered to a subject, such as a subject with a disease or condition or at risk of developing a disease or condition. In some aspects, the methods thereby treat, for example, by alleviating one or more symptoms of a disease or condition, such as by reducing the tumor burden in cancers expressing antigens recognized by engineered cells (e.g., engineered T cells).

[0299] Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T-cell therapy is described in the following literature: US2003 / 0170238; US Patent No. 4,690,915; Rosenberg, “Cell transfer immunotherapy for metastatic solid cancer—what clinicians need to know.” Nature reviews Clinicaloncology 8.10(2011):577; Themeli et al., “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.” Nature biotechnology 31.10(2013):928; Tsukahara et al., “CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models.” Biochemical and biophysical research communications 438.1(2013):84-89; Davila et al., “CD19 CAR-targeted T cells induce long-term remission and B-cell aplasia in an immunocompetent mouse model of B-cell aplasia.” Cell acute lymphoblastic leukemia.” PloS one 8.4 (2013); each of these articles is incorporated into this paper in its entirety by reference.

[0300] In some implementations, cell therapy, such as adoptive T-cell therapy, is performed via autologous transfer, wherein the T cells are isolated from and / or otherwise prepared from a subject to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, engineered cells are derived from a subject requiring treatment, such as a patient, and the engineered cells are administered to the same subject after isolation and processing.

[0301] In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed via allogeneic transfer, wherein the T cells are isolated and / or otherwise prepared from a subject other than the subject to receive or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the engineered cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0302] In some implementations, the subject's HLA class or HLA supertype is identified. In some implementations, the subject is treated with a cell therapy containing antigens in a background that can identify the HLA class or HLA supertype.

[0303] In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or condition, such as a tumor, prior to the administration of engineered cells or a composition containing engineered cells. In some aspects, the subject is refractory or unresponsive to other therapeutic agents. In some embodiments, the subject has a persistent or recurrent disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, the subject is effectively treated despite resistance to another therapy.

[0304] In some implementations, the subject responds to other therapeutic agents and treatment with those agents reduces the disease burden. In some aspects, the subject initially responds to the therapeutic agent but exhibits a relapse of the disease or condition over time. In some implementations, the subject does not experience a relapse. In some such implementations, the subject is identified as being at risk of relapse, such as being at high risk of relapse, and therefore engineered cells are administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse. In some implementations, the subject has not received prior treatment with other therapeutic agents.

[0305] In some embodiments, engineered cells are administered at a desired dose, which in some aspects includes a dose or number of desired cells or cell types and / or a desired ratio of cell types. Thus, in some embodiments, the cell dose is based on the total number of cells (or the number per kg of body weight) and the desired ratio of a single population or subtype, such as the CD4+ to CD8+ ratio. In some embodiments, the cell dose is based on the desired total number of cells (or the number per kg of body weight) within a single population or single cell type. In some embodiments, the dose is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells within a single population.

[0306] In some embodiments, cell populations or subtypes, such as CD8+ and CD4+ T cells, are administered at a desired dose of total cells (such as a desired dose of T cells) or within permissible variation thereof. In some embodiments, the desired dose is a desired number of cells or a desired number of cells per unit body weight of the subject to which the engineered cells are administered, e.g., cells / kg. In some embodiments, the desired dose is equal to or greater than a minimum number of cells or a minimum number of cells per unit body weight. In some embodiments, in the total cells administered at the desired dose, a single population or subtype is present at or near a desired output ratio (such as the ratio of CD4+ to CD8+), e.g., within a specific permissible variation or error of such a ratio.

[0307] In some embodiments, engineered cells are administered at a desired dose (such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells) or within permissible variations for one or more individual cell populations or subtypes. In some embodiments, the desired dose is the desired number of cells of the subtype or population, or the desired number of such cells per unit body weight of the subject receiving the engineered cells, e.g., cells / kg. In some embodiments, the desired dose is equal to or greater than the minimum number of cells of the population or subtype, or the minimum number of cells of the population or subtype per unit body weight.

[0308] Therefore, in some embodiments, the dose is based on the expected fixed dose and expected proportion of total cells, and / or based on the expected fixed dose of one or more (e.g., each) single subtype or subpopulation. Therefore, in some embodiments, the dose is based on the expected fixed dose or minimum dose of T cells, and the expected ratio of CD4+ to CD8+ cells, and / or based on the expected fixed dose or minimum dose of CD4+ and / or CD8+ cells.

[0309] In some implementations, engineered cells are administered to the subject in the range of approximately 1 million to approximately 100 billion cells, such as, for example, 1 million to approximately 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the above values), such as approximately 10 million to approximately 100 billion cells (e.g., approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately...). 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the above values), and in some cases, approximately 100 million cells to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells) or any value between these ranges.

[0310] In some implementations, the dose of total cells and / or the dose of individual cell subsets is equal to or about 10. 4 and equal to or approximately 10 9 Within the range of cells per kilogram (kg) of body weight, such as in 10 5 With 10 6 Between cells per kilogram of body weight, for example, at least or at least about or equal to or about 1 × 10⁻⁶. 5 cells / kg, 1.5 × 10 5 Cells / kg, 2×10 5 cells / kg, or 1×10 6 Cells per kilogram of body weight. For example, in some embodiments, engineered cells are administered at doses equal to or approximately 10. 4 and equal to or approximately 10 9 Between 10 T cells / kg body weight, such as in 10 5 With 10 6 Between 1 T cells / kg body weight, for example, at least or at least about or equal to or about 1 × 10 5 1.5 × 10 T cells / kg 5 T cells / kg, 2×10 5 T cells / kg, or 1×10 6 T cells per kilogram of body weight.

[0311] In some implementations, engineered cells are administered at doses equal to or about 10 ppm or within a specific error range of the following dosages: 4 and equal to or approximately 10 9 Between 10 CD4+ and / or CD8+ cells per kilogram (kg) of body weight, such as in 10 5 With 10 6 Between 1 CD4+ and / or CD8+ cells per kilogram of body weight, for example, at least or at least about or equal to or about 1 × 10⁻⁶ cells per kilogram of body weight. 5 CD4+ and / or CD8+ cells / kg, 1.5 × 10⁻⁶ 5 CD4+ and / or CD8+ cells / kg, 2×10 5 CD4+ and / or CD8+ cells / kg, or 1×102 6 CD4+ and / or CD8+ cells per kilogram of body weight.

[0312] In some implementations, the engineered cells are administered at doses higher than and / or at least about 1 × 10⁻⁶. 6 Approximately 2.5 × 10 6 Approximately 5×10 6 Approximately 7.5 × 10 6 or approximately 9×10 6 100 CD4+ cells, and / or at least about 1 × 10⁶ cells. 6 Approximately 2.5 × 10 6 Approximately 5×10 6 Approximately 7.5 × 10 6 or approximately 9×10 6 100 CD8+ cells, and / or at least about 1000 CD8+ cells. 6 Approximately 2.5 × 10 6 Approximately 5×10 6 Approximately 7.5 × 10 6 or approximately 9×10 6 T cells. In some implementations, the engineered cells are administered at the following dose or within a specific error range of the following dose: approximately 10 8 With 10 12 Between T cells or about 10 10 With 10 11 Between each T cell, approximately 10 8 With 10 12 Between CD4+ cells or about 10 10 With 10 11 Between CD4+ cells, and / or approximately 10 8 With 10 12 Between CD8+ cells or about 10 10 With 10 11Between CD8+ cells.

[0313] In some implementations, engineered cells are applied at a desired output ratio of multiple cell populations or subtypes (such as CD4+ and CD8+ cells or subtypes) or within an acceptable range of said desired output ratio. In some aspects, the desired ratio may be a specific ratio or a range of ratios. For example, in some implementations, the desired ratio (e.g., the ratio of CD4+ cells to CD8+ cells) is between 1:5 and 5:1 (or greater than 1:5 and less than 5:1), or between 1:3 and 3:1 (or greater than 1:3 and less than 3:1), such as between 2:1 and 1:5 (or greater than 1:5 and less than 2:1), such as 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1 The ratios are 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some respects, the permissible difference is within approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% of the desired ratio, including any value between these ranges. In some respects, the TCRs described herein provide improved expression and activity, thereby providing therapeutic effects even at low effector:target (E:T) ratios.

[0314] Optimal response to a therapy may depend on the ability of engineered recombinant receptors, such as TCRs, to be consistently and reliably expressed on the surface of engineered cells and / or to bind target antigens. For example, in some cases, the nature of certain recombinant receptors (e.g., TCRs) can affect their expression and / or activity, especially when expressed in cells used in cell therapy, such as human T cells. In some cases, the expression level of a particular recombinant receptor (e.g., TCR) may be low, and the activity of engineered cells (such as human T cells) expressing such recombinant receptors may be limited due to poor expression or poor signaling activity. In some cases, the consistency and / or efficiency of recombinant receptor expression, and the receptor's activity, are limited in certain cells or cell populations where the therapeutic approach is available. In some cases, a large number of engineered cells (e.g., engineered T cells) (with a high effector:target (E:T) ratio) are required to exhibit functional activity. In some embodiments, the desired ratio (E:T ratio) is between 1:10 and 10:1 (or greater than 1:10 and less than 10:1), or between 1:1 and 10:1 (or greater than 1:1 and less than 5:1), such as between 2:1 and 10:1. In some embodiments, the E:T ratio is greater than or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0315] For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the engineered cells are administered for preventive or therapeutic purposes, prior treatment, the subject's clinical history and response to the engineered cells, and the judgment of the attending physician. In some embodiments, the composition and cells are appropriately administered to the subject in a single dose or as part of a series of treatments.

[0316] The engineered cells described herein can be administered by any suitable method, such as by bolus infusion, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjunctival injection, subconjuntival injection, sub-Tenon's injection, retroocular injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, they are administered via parenteral, intrapulmonary, and intranasal administration, and if local treatment is required, via intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus injection of engineered cells. In some embodiments, engineered cells are administered by, for example, multiple bolus injections over a period not exceeding 3 days, or by continuous infusion.

[0317] In some embodiments, engineered cells are administered as part of a combination therapy, such as simultaneously with or sequentially with another therapeutic intervention (such as an antibody or engineered cell or receptor or agent, such as a cytotoxic agent or therapeutic agent). In some embodiments, engineered cells are co-administered with or in combination with another therapeutic intervention, said administration being simultaneous or sequentially in any order. In some cases, engineered cells are co-administered with another therapy at sufficiently close proximity in time such that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, engineered cells are administered prior to one or more additional therapeutic agents. In some embodiments, engineered cells are administered after one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents include cytokines, such as IL-2, for example, to enhance persistence. In some embodiments, the method includes the administration of a chemotherapeutic agent.

[0318] Following the administration of engineered cells, the bioactivity of the engineered cell population in some embodiments is measured, for example, by any of a variety of known methods. Parameters to be evaluated include the specific binding of the engineered cells (e.g., engineered T cells) to the antigen in vivo (e.g., by imaging) or in vitro (e.g., by ELISA or flow cytometry). In some embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in the following literature: for example, Kochenderfer et al., “Construction and pre-clinicale evaluation of an anti-CD19 chimeric antigen receptor.” Journal of Immunotherapy (Hagerstown, Md.: 1997) 32.7 (2009): 689 and Hermans et al., “The VITAL assay: a versatile fluorometric technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.” Journal of Immunological Methods 285.1 (2004): 25-40. In some implementations, the bioactivity of engineered cells is measured by determining the expression and / or secretion of one or more cytokines such as CD107a, IFNγ, IL-2, and TNF. In other aspects, bioactivity is measured by assessing clinical outcomes such as a reduction in tumor burden or load.

[0319] Example

[0320] In the following sections, several embodiments are provided to further describe the invention as covered by the aspects of this disclosure as described above. It should be noted that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of this disclosure.

[0321] Example 1. Design of several Aspire-TCR subunits

[0322] In this embodiment, several designs for the Aspire-TCR subunit are provided.

[0323] 1.1. Design of the engineered CD3 zeta subunit

[0324] Figure 2AThe structure of an engineered CD3 zeta (i.e., CD3z) subunit according to some embodiments of this disclosure is shown, the subunit being constructed essentially by fusing a co-stimulatory region (e.g., a co-stimulatory domain of CD28, 4-1BB, etc.) with the C-terminus of a CD3z assembly (e.g., human CD3z). Flexible connectors (in...) Figure 2A (Seen in a straight line) can be arranged between the CD3z component and the co-stimulatory region, but this is optional. It should be noted that, according to some other embodiments of the engineered CD3z subunit (not shown), the co-stimulatory region may be within the intracellular domain of the CD3z component, and for example, between the transmembrane domain and the intracellular domain of the CD3z component. Such engineered CD3z subunits can be ectopically expressed in immune cells to improve certain properties of immune cells, such as enhanced proliferative capacity.

[0325] 1.2. Design of the engineered CD3 epsilon subunit

[0326] Figure 2B The structure of an engineered CD3 epsilon (i.e., CD3e) subunit is shown, which substantially includes a target recognition region (e.g., scFv, sdAb, ligand, etc.) fused to the N-terminus of a truncated CD3 epsilon (i.e., "ΔCD3e", which lacks the intracellular domain corresponding to amino acids 151-179 of human CD3e (SEQ ID NO:5)). According to different embodiments of the engineered CD3e subunit as provided herein, one or more functional regions (e.g., CD40 motif, 4-1BB motif, LAT motif, etc.; such as...) Figure 2B The area shown in the dashed box can be optionally further fused to the C-terminus of the truncated CD3e, but some embodiments of the engineered CD3e subunit as provided herein may not include other functional regions (i.e., the C-terminus of the truncated CD3epsilon subunit “ΔCD3e” is the C-terminus of the engineered CD3e subunit). Flexible joints (such as those made by...) can optionally be arranged between the target recognition region and the truncated CD3e, and / or between the truncated CD3e and one or more functional regions. Figure 2B (as shown by the straight line in the diagram), but not required. Any embodiment of the engineered CD3e subunit as described above can be used alone or in conjunction with, for example, Figure 2A The engineered CD3z subunits shown and described above are used in combination to engineer immune cells.

[0327] 1.3. Engineering Design of Ligand-Based Aspire-TCR Subunits

[0328] Figure 2CStructures of a series of embodiments of ligand-based Aspire-TCR subunits are shown, said subunits substantially comprising subunits fused to a TCR signaling complex subunit (e.g., one of a TCR alpha subunit, a TCR beta subunit, a CD3gamma subunit, a CD3 delta subunit, or a CD3 epsilon subunit); Figure 2C The ligand portion (“ligand”; e.g., IL13(E13Y), GM-CSF, etc.) is the N-terminal portion of the TCR signaling complex subunit (represented as “TCRa / TCRb / CD3g / CD3d / CD3e”). In each of these embodiments, the ligand portion is essentially a target recognition portion that specifically recognizes the corresponding homologous receptor expressed on the target cell. A flexible linker, such as a GS linker, may optionally be disposed between the ligand portion and the TCR signaling complex subunit. It should be noted that, in addition to being fused to the N-terminus of the TCR complex subunit, the ligand portion may be inserted at any other location within the extracellular domain of the TCR signaling complex subunit. Upon expression in immune cells, the engineered target-recognition ligand portion of the TCR complex subunit can guide such engineered immune cells to specifically recognize target cells expressing the homologous ligand receptor, thereby enabling the immune cells to exert their cytotoxicity against the target cells. Any embodiment of the engineered ligand-based Aspire-TCR subunit as described above can be used alone or in conjunction with... Figure 2A The engineered CD3z subunit combination shown is used to engineer immune cells.

[0329] Example 2. Expression and characterization of IL13Ra2-targeted Aspire-TCR subunits in T cells based on ligands.

[0330] In this embodiment, a ligand-based Aspire-TCR subunit is provided, which substantially encodes the fusion protein “IL13(E13Y)-CD3e”, the fusion protein comprising an “IL13(E13Y)” ligand moiety (SEQ ID NO:4) fused to the N-terminus of the human CD3epsilon subunit (SEQ ID NO:5) via a GS linker (SEQ ID NO:6). The “IL13(E13Y)” ligand moiety is designed to specifically target IL-13Rα2 (or IL13Ra2), IL-13Rα2 being a recognized cell surface marker for glioma and renal cell carcinoma (RCC). More specifically, the “IL13(E13Y)” ligand moiety comprises a mutant form of the IL-13 cytokine, IL-13(E13Y), which has shown selective affinity for IL-13Rα2 and is unlikely to bind to the IL-13Rα1 / IL-4β / γc receptor in normal tissues. In addition, IL13 (E13Y) ligands have been used in IL-13Rα2-CAR-T cell therapy for the treatment of gliomas, as described in detail in U.S. Patent No. 7,514,537B, the disclosure of which is incorporated herein by reference in its entirety.

[0331] 2.1. Design of Four Aspire-TCR Constructs Targeting IL13Ra2

[0332] like Figure 3 As shown, four Aspire-TCR constructs were designed, including "CD3e", "BBe", "CD3e-BBz", and "CD3e-28z". Descriptions of these four constructs are provided below:

[0333] (1) "CD3e" construct: encodes the fusion protein "IL13(E13Y)-CD3e" as described above.

[0334] (2) “BBe” construct: encodes fusion protein “IL13(E13Y)-CD3e-41BB”, wherein the C-terminus of “IL13(E13Y)-CD3e” is further fused to the 4-1BB intracellular domain (“41BB”; SEQ ID NO:3) via a linker (“linker”; SEQ ID NO:7).

[0335] (3) The “CD3e-BBz” construct: encodes two fusion proteins, “IL13(E13Y)-CD3e” and “CD3z-41BB”, separated by a 2A self-cleaving peptide T2A (SEQ ID NO:8). “CD3z-41BB” (i.e., “BBz”) is essentially… Figure 2AOne embodiment of the engineered CD3z subunit shown is wherein the C-terminus of the human CD3z subunit (SEQ ID NO:1) is fused with the human 4-1BB intracellular domain (SEQ ID NO:3).

[0336] (4) The “CD3e-28z” construct: encodes two fusion proteins, “IL13(E13Y)-CD3e” and “CD3z-CD28”, separated by the 2A self-cleaving peptide T2A (SEQ ID NO:8). “CD3z-CD28” (i.e., “28z”) is... Figure 2A Another embodiment of the engineered CD3z subunit shown is wherein the C-terminus of the human CD3z subunit (SEQ ID NO:1) is fused with the human CD28 co-stimulatory region (SEQ ID NO:2).

[0337] In the “CD3e-BBz” and “CD3e-28z” constructs, the IL13(E13Y)-CD3e and engineered CD3z subunits can be expressed independently when the T2A peptide undergoes self-cleavage.

[0338] 2.2. Expression of four IL13(E13Y) Aspire-TCR constructs in T cells

[0339] First, the in vitro expression of the four constructs described above in T cells was examined. In short, primary human T cells were transduced with genetic constructs encoding the four Aspire-TCR constructs and expanded for 12 days in the presence of IL-2. T cells expressing the Aspire-TCR constructs were named "Aspire-T" cells. After expansion, IL13 expression in these IL13(E13Y) Aspire-T cells was measured by flow cytometry, representing the expression of the Aspire-TCR subunit containing the IL13(E13Y) ligand. Figures 4A-4E As shown, compared with non-transduced (“NT”) T cells ( Figure 4A Compared to the four Aspire-TCR constructs "CD3e", "BBe", "CD3e-28z", and "CD3e-BBz", the four Aspire-TCR constructs "CD3e", "BBe", "CD3e-28z", and "CD3e-BBz" are more advanced. Figure 4B-4E Each of the elements in ) can be expressed effectively.

[0340] 2.3. Cytotoxicity and activation of IL13 (E13Y) Aspire-T cells against target tumor cells

[0341] The cytotoxicity of IL13(E13Y) Aspire-T cells against target tumor cells was further investigated. Briefly, different Aspire-T cells were individually cultured overnight with CFSE-prelabeled tumor cells (specifically the U251 MG glioma cell line as the target tumor cells). Subsequently, tumor cells were harvested for viability assessment via 7-AAD. Figure 5 As shown, compared to the “NT” T cell control, the three IL13(E13Y) Aspire-T cell lines, “CD3e,” “CD3e-28z,” and “CD3e-BBz,” exhibited significant cytotoxicity against U251 glioblastoma cells expressing IL13Ra2. Furthermore, compared to “CD3e” Aspire-T cells expressing only the engineered target recognition subunit “IL13(E13Y)-CD3e,” the “CD3e-28z” and “CD3e-BBz” Aspire-T cells, characterized by the engineered CD3z subunit (except for the engineered target recognition subunit “IL13(E13Y)-CD3e,” showed significantly enhanced cytotoxicity against target tumor cells. This observation suggests that armoring T cells expressing engineered target recognition TCR subunits with the engineered CD3z subunit can enhance cytotoxicity against target tumor cells.

[0342] To evaluate antigen-specific stimulation of various Aspire-T cells, the following assays were performed. Briefly, different Aspire-T cells were cultured for 48 hours with or without U251 target tumor cells. Subsequently, the supernatant was collected, and IFN-γ levels were measured by ELISA. Figure 6As shown, in the presence of target tumor cells (i.e., the "U251" group), "NT" T cells were not activated by the antigen, while the three IL13(E13Y) Aspire-T cell types "CD3e", "CD3e-28z", and "CD3e-BBz" all showed a significant increase in IFN-γ levels, indicating significant antigen activation. However, it is noteworthy that in the absence of target tumor cells (i.e., the "no-target" group), "CD3e" T cells (i.e., T cells expressing only the engineered target recognition subunit "IL13(E13Y)-CD3e") showed relatively high background activation (i.e., activation in the absence of antigen stimulation), which was approximately 3-10 times higher than in the other groups. Specifically, “CD3e-28z” T cells (i.e., T cells co-expressing the engineered target recognition subunit IL13(E13Y)-CD3e and the engineered “CD3z-CD28” subunit) showed almost no activation (i.e., levels comparable to control “NT” T cells) without antigen stimulation, while “CD3e-BBz” T cells (i.e., T cells co-expressing the engineered target recognition subunit IL13(E13Y)-CD3e and the engineered “CD3z-4-1BB” subunit) showed slightly higher background activation than “CD3e-28z” T cells, but still much lower than “CD3e” T cells (i.e., only about 1 / 5 of “CD3e” T cells). These results suggest that armoring T cells expressing engineered target recognition TCR subunits with engineered CD3z subunits can reduce unwanted background activation.

[0343] 2.4. Cytotoxicity and activation of IL13 (E13Y) Aspire-T cells against non-target tumor cells

[0344] To further examine the specificity of the four Aspire-T cell types, their cytotoxicity and activation against non-target cells were also investigated.

[0345] First, the cytotoxicity of T cells against non-target cells was examined, and the non-target tumor cell line THP-1, which expresses IL13Ra1 instead of IL13Ra2, was tested (see [link to T cell cytotoxicity test]). Figures 7A-7B ).like Figure 7CThe results showed that both "CD3e" (i.e., T cells expressing only the engineered target recognition subunit "IL13(E13Y)-CD3e") and "BBe" (i.e., T cells expressing the engineered target recognition subunit "IL13(E13Y)-CD3e-4-1BB") Aspire-T cells exhibited relatively high cytotoxicity against non-target THP-1 cells, while "CD3e-28z" (i.e., T cells co-expressing the engineered target recognition subunits "IL13(E13Y)-CD3e" and the engineered "CD3z-CD28" subunit) and "CD3e-BBz" (i.e., T cells co-expressing the engineered target recognition subunits "IL13(E13Y)-CD3e" and the engineered "CD3z-4-1BB") Aspire-T cells exhibited much lower cytotoxicity, essentially similar to the cytotoxicity of "NT" control T cells.

[0346] Furthermore, the activation of T cells targeting non-target THP-1 cells was further investigated. For example... Figure 8A The results showed that both "CD3e" and "BBe" Aspire-T cells exhibited increased activation upon contact with non-target THP-1 cells, as evidenced by the increased percentage of IFN-γ-producing cells in the presence of THP-1 cells (the "THP-1 group") compared to the absence of THP-1 cells (the "no-target" group). In stark contrast, "CD3e-28z" Aspire-T cells showed no activation. Further analysis of IFN-γ secretion levels... Figure 8B In the independent experiments shown, “CD3e” Aspire-T cells showed significant activation upon contact with non-target THP-1 cells, while “CD3e-28z” Aspire-T cells showed only slight activation.

[0347] Figures 7A-7C and Figures 8A-8B The results above indicate that armoring T cells expressing engineered target recognition TCR subunits with engineered CD3z subunits can significantly reduce unwanted activation and cytotoxicity against non-target cells.

[0348] In summary, Aspire-T cells expressing only the ligand moiety fused with IL13 (E13Y) exhibit cytotoxicity against tumor cells expressing IL13Ra2 (target tumor cells). Figure 5 However, these Aspire-T cells exhibit a certain level of background activation even in the absence of antigens, or can even be activated in the presence of non-target cells, thereby causing cytotoxicity against non-target cells. Figure 7CNevertheless, the limited level of specificity of these Aspire-T cells can be rescued by co-expression of the engineered CD3z subunit provided herein (i.e., CD3z fused with the CD28 or 4-1BB co-stimulatory domain), which can significantly reduce background activation of engineered T cells in the absence of the antigen (see [link to article]). Figure 6 It significantly reduces unwanted cytotoxicity against non-target cells (see [link]). Figure 7C , 8A And 8B), and further enhance cytotoxicity against target tumor cells (see 8B). Figure 5 This provides a more favorable and safer therapeutic profile.

[0349] Example 3. Expression and characterization of ScFv-based ALPP-targeted Aspire-TCR subunits in T cells

[0350] In this embodiment, an Aspire-TCR subunit based on scFv is provided, which substantially comprises a fusion protein “F8-CD3e”, wherein the fusion protein comprises an anti-ALPP scFv “F8” (SEQ ID NO:9) fused to the N-terminus of a human CD3 epsilon subunit (SEQ ID NO:5) via a GS linker (SEQ ID NO:6). This Aspire-TCR is specifically designed to target ALPP (alkaline phosphatase, placenta; also known as placental alkaline phosphatase or PLAP), which has been established as a specific tumor marker for ovarian cancers (e.g., ovarian adenocarcinoma, serous cystadenocarcinoma, undifferentiated carcinoma, and dysgerminoma) and seminoma.

[0351] 3.1. Design of two Aspire-TCR constructs targeting ALPP

[0352] Specifically, two types of constructs were designed, and their structural diagrams are shown in... Figure 9 As shown in the diagram. These two constructs can be individually cloned into a pMP71 retroviral vector to generate engineered T cells. More specifically, these two constructs include:

[0353] (1) The “F8-BBe” construct essentially encodes the fusion protein “F8-CD3e-4-1BB”, wherein the C-terminus of the fusion protein “F8-CD3e” as described above is further fused to the intracellular domain of 4-1BB (“4-1BB”; SEQ ID NO:3) via a linker (“linker”; SEQ ID NO:7);

[0354] (3) The “F8-28z” construct essentially encodes two fusion proteins, “F8-CD3e” and “CD3z-CD28”, separated by a 2A self-cleaving peptide T2A (SEQ ID NO:8). In this paper, “CD3z-CD28” is the same as described in Example 2 above, wherein the C-terminus of the human CD3z subunit (SEQ ID NO:1) is fused to the human CD28 co-stimulatory region (SEQ ID NO:2). 3.2. Expression of the anti-ALPP Aspire-TCR construct in T cells

[0355] Using non-transduced (“NT”) T cells as a negative control, the in vitro expression of the two Aspire-TCR constructs in T cells was examined. Briefly, primary human T cells were transduced with different constructs and expanded for 5–6 days in the presence of IL-2. After expansion, F8 scFv expression was measured by flow cytometry. Figures 10A-10C As shown, both Aspire-TCR constructs can be effectively expressed in transduced T cells.

[0356] 3.3. Cytotoxicity and activation of anti-ALPP Aspire-T cells against target tumor cells

[0357] The cytotoxicity of anti-ALPP Aspire-T cells against target tumor cells was further investigated. Briefly, different Aspire-T cells were cultured overnight with CFSE-prelabeled tumor cells (specifically SiHa or Caski cells as target tumor cells). Subsequently, tumor cells were collected for viability assessment via 7-AAD. Figure 11A-11B As shown, compared with the "NT" control T cells, the "F8-28z" and "F8-BBe" Aspire-T cells respectively showed a significant effect on SiHa cells ( Figure 11A ) and Caski cells ( Figure 11B Both showed significant cytotoxicity.

[0358] To examine antigen-specific stimulation of various Aspire-T cells, Aspire-T cells were cultured for 48 hours with or without target tumor cells (SiHa or Caski). The supernatant was then collected, and IFN-γ levels were measured by ELISA. Results were... Figure 12As shown in the figure, control “NT” T cells did not show activation in the “no target” group or in any targeted tumor group (SiHa and Caski tumor cells). Both “F8-28z” and “F8-BBe” Aspire-T cells showed antigen-specific activation, as evidenced by increased IFN-γ secretion in the presence of target tumor cells (with antigen) compared to the absence of antigen (“no target” group). “F8-BBe” Aspire-T cells (expressing only the target recognition “F8-CD3e-4-1BB” subunit) showed a relatively high level of background activation in the absence of antigen, while “F8-28z” Aspire-T cells (co-expressing the target recognition “F8-CD3e” subunit and the engineered “CD3z-CD28” subunit) showed only minimal background activation in the absence of antigen. These results suggest that armoring T cells expressing engineered target recognition TCR subunits with the engineered CD3z subunit can reduce unwanted background activation. Although both “F8-28z” and “F8-BBe” Aspire-T cells showed good antigen-specific activation, “F8-28z” T cells showed favorable minimal background activation in the absence of antigen stimulation.

[0359] 3.4. Cytotoxicity and activation of anti-ALPP Aspire-T cells against non-target tumor cells

[0360] To further examine the specificity of the two anti-ALPP Aspire-T cell types, their cytotoxicity against non-target cells was further investigated. Figures 13A-13B As shown, target SiHa cells that tested positive for the antigen ALPP (as shown) Figure 13B In contrast, A549 cells are non-target tumor cells that tested negative for the antigen ALPP. Figure 13A Therefore, A549 cells were used to evaluate the non-specific cytotoxicity of two anti-ALPP Aspire-T cell lines. Figure 13C The results showed that "F8-BBe" T cells (expressing only the target recognition "F8-CD3e-4-1BB" subunit) exhibited relatively high cytotoxicity against non-target A549 cells. Conversely, "F8-28z" T cells (co-expressing the target recognition "F8-CD3e" subunit and engineered "CD3z-CD28" subunit) showed much lower off-target cytotoxicity, essentially similar to the cytotoxicity of control "NT" T cells.

[0361] Therefore, similar to Example 2 described above, although anti-ALPP Aspire-T cells “F8-BBe” (expressing only the target-recognizing “F8-CD3e-4-1BB” subunit) exhibited cytotoxicity against SiHa or Caski target tumor cells ( Figure 11A-11B However, they also exhibit relatively high levels of background activation in the absence of antigens. Figure 12 ), and showed relatively high levels of off-target cytotoxicity against non-target tumor cells. Figure 13C The limited specificity of “F8-BBe” Aspire-T cells can be significantly improved or even rescued by co-expression of the engineered “CD3z-CD28” subunit. This co-expression of both the anti-ALPP TCR subunit and the engineered CD3z-CD28 subunit in “F8-28z” Aspire-T cells significantly reduced background activation in the absence of the antigen. Figure 12 It also reduced off-target cytotoxicity against non-target cells. Figure 13C This provides a more favorable and safer treatment profile.

[0362] Example 4. Expression and characterization of the truncated CD3e-based Aspire-TCR subunit in T cells.

[0363] This embodiment essentially provides a method according to the description in Embodiment 1 above and in... Figure 2B The diagram illustrates a series of engineered CD3 epsilon (i.e., CD3e) subunits based on truncated CD3e, representing different embodiments of the engineered CD3e subunits. These engineered CD3e subunits all include a target recognition region fused to the N-terminus of a truncated CD3 epsilon (“ΔCD3e” (SEQ ID NO:10), which lacks the C-terminal 29 amino acid residues compared to the full-length CD3e subunit (SEQ ID NO:5)), thereby forming a fusion peptide “target recognition region-ΔCD3e”. Depending on the embodiment, one or more functional regions (e.g., CD40 motif, 4-1BB motif, LAT motif, etc.) may be optionally further fused to the C-terminus of the fusion peptide “target recognition region-ΔCD3e”.

[0364] In this embodiment, the anti-ALPP scFv is used as a target recognition region for the engineered CD3e subunit, and it can be F8scFv (SEQ ID NO:9) or A02 scFv (SEQ ID NO:30, disclosed in US20220125845A1, the contents of which are incorporated herein by reference in their entirety). F8 scFv is used for... Figure 16A-23B All in vitro experiments, and A02 scFv for Figure 25-27 In vivo experiments.

[0365] 4.1. Design of the Aspire-TCR Construct

[0366] like Figure 14 As shown in Table 1, a total of 11 Aspire-TCR constructs AK were designed. Each construct encodes two chimeric proteins separated by a T2A linker (SEQ ID NO:8), including an engineered CD3e subunit and an engineered CD3z subunit. Each of these constructs can be cloned into a pMP71 retroviral vector to generate Aspire-T cells.

[0367] In this document, the engineered CD3e subunits in each construct are essentially target-recognizing Aspire-TCR subunits, comprising scFvs fused to a full-length CD3e subunit (see Design A) or fused to a truncated CD3e (“ΔCD3e”) subunit (see Design BK, which has different domains / motifs / regions in each different embodiment) via a flexible linker (e.g., a GS linker (SEQ ID NO: 6)). The engineered “CD3z-CD28” subunit or “28z” comprises a CD3z or modified CD3z subunit, wherein the intracellular domain further comprises a CD28 co-stimulatory domain “CD28”. In the following examples, both engineered CD3 subunits are co-expressed in T cells and characterized in vitro and in vivo. It should be noted that Design A is substantially the same as the “F8-CD3e” construct in Example 3 above, which is primarily used in this example as a control for characterizing Design BK.

[0368] The following domains / motifs / regions may be included in the design: (1) “scFv”; (2) human CD3e component: “CD3e” (SEQ ID NO:5), or “ΔCD3e” (i.e., CD3e whose intracellular region is truncated; SEQ ID NO:10); (3) human CD3z signal transduction domain: “CD3z ITAM2-3” (SEQ ID NO:12), “CD3z ITAM3” (SEQ ID NO:11), “CD3z-LHYRHQMQ” (SEQ ID NO:20), “CD3z ITAM2-3-LHYLSLMQ” (SEQ ID NO:18), or “CD3z ITAM3-LHYLSLMQ” (SEQ ID NO:19); (4) human 4-1BB functional region: “4-1BB motif” (SEQ ID NO:15) or “4-1BB” (abbreviated as “4-1BB intracellular domain”; SEQ ID NO:3); or CD28 costimulatory domain (“CD28”; SEQ ID NO:3). (SEQ ID NO:2); and (5) different modified regions, domains or motifs designed to enhance Aspire-T cell function: “FcεR1γ” (i.e., the intracellular domain of FcεR1γ; SEQ ID NO:25); “OX40” (i.e., the intracellular domain of OX40; SEQ ID NO:26); “FceR1g+OX40” (i.e., a complex functional region formed by fusing “FcεR1γ” and “OX40” from the N-terminus to the C-terminus; SEQ ID NO:13); “CD40” (i.e., the intracellular domain of CD40; SEQ ID NO:27); “DAP2” (i.e., the intracellular domain of DAP2; SEQ ID NO:28); “CD40+DAP12” (i.e., a complex functional region formed by fusing “CD40” and “DAP2” from the N-terminus to the C-terminus; SEQ ID NO:14); “CD40 motif” (SEQ ID NO:17); and “LAT motif” (SEQ ID NO:16). The table below summarizes the structures of these 11 Aspire-TCR builds (design AK).

[0369] Table 1: Structures of 11 Aspire-TCR constructs (AK)

[0370] design Construct Engineered CD3e subunit Engineered CD3z subunit [A] scFv-CD3e-28z scFv-CD3e 28z [B] scFv-ΔCD3e-28z scFv-ΔCD3e 28z [C] scFv-ΔCD3eZ-28z scFv-ΔCD3eZ 28z [D] scFv-ΔCD3e-FOZ-28z scFv-ΔCD3e-FOZ 28z [E] scFv-ΔCD3e-40DZ-28z scFv-ΔCD3e-40DZ 28z [F] scFv-ΔCD3e-41BBMZ-28z scFv-ΔCD3e-41BBMZ 28z [G] scFv-ΔCD3e-41BBM-M1Z-28z scFv-ΔCD3e-41BBM-M1Z 28z [H] scFv-ΔCD3e-CD40M-M1Z-28z scFv-ΔCD3e-CD40M-M1Z 28z [I] scFv-ΔCD3e-28z-S3 scFv-ΔCD3e 28z-S3 [J] scFv-ΔCD3e-S5-28z-S3 scFv-ΔCD3e-S5 28z-S3 [K] scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 scFv-ΔCD3e-41BBM-M1Z-S5 28z-S3

[0371] like Figure 15A-15K As shown, by Figure 14Each of the 11 Aspire-TCR constructs shown encodes an engineered target that recognizes the CD3e subunit and the engineered CD3z subunit. When expressed in immune cells, these subunits can be incorporated into the T-cell receptor signaling complex, thereby forming the Aspire-TCR complex.

[0372] 4.2. In vitro expression of the Aspire-TCR construct in T cells

[0373] The in vitro expression of these constructs in T cells was examined. In short, primary human T cells were transduced with different constructs and expanded for 12-13 days in the presence of IL-2. After expansion, scFv expression on different Aspire-T cells was measured by flow cytometry. Figure 16A-16D As shown in 17A-17F and 18A-18F, and scFv-CD3e-28z ( Figure 14 Compared to design A), all Aspire-TCRs containing “ΔCD3e” (design BK) showed unexpectedly better expression in primary T cells, indicating that the C-terminal 29-amino acid (AA) portion of CD3e negatively affects the expression level of engineered CD3e subunits, and that removing the 29-AA portion from the CD3e component significantly enhances the expression of engineered CD3e subunits.

[0374] 4.3. In vitro expansion capacity and post-expansion memory phenotype analysis of Aspire-T cells

[0375] The in vitro expansion capacity of scFv Aspire-T cells was investigated as described below. Primary human T cells were used... Figure 14 A single scFv Aspire-TCR construct was transduced and expanded for 12-13 days in the presence of IL-2. The fold increase was calculated upon cell harvest. Figures 19A-19C As shown, all Aspire-T cells with "ΔCD3e" (design BK) expanded well in culture; however, the expansion folds varied. Generally, control untransduced ("NT") T cells showed the best expansion capacity compared to all transduced Aspire-T cells. scFv-ΔCD3e-28z (design B) and scFv-ΔCD3eZ-28z (design C) showed similar T cell expansion levels compared to T cells expressing scFv-CD3e-28z (design A). Figure 19AWhen compared to T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-FOZ-28z (design D) or scFv-ΔCD3e-41BBZ-28z (design F) showed slightly lower expansion levels, and T cells expressing scFv-ΔCD3e-40DZ-28z (design E) showed much lower expansion levels. Figure 19B Furthermore, compared to T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-41BBM-M1Z-28z (design G), scFv-ΔCD3e-28z-S3 (design I), or scFv-ΔCD3e-S5-28z-S3 (design J) showed similar levels of proliferative capacity, while T cells expressing scFv-ΔCD3e-CD40M-M1Z-28z (design H) proliferated more slowly in culture, and T cells expressing scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) proliferated more rapidly in culture. Figure 19C ).

[0376] Furthermore, the proportion of memory T cells in different Aspire-T cell types after expansion was investigated as follows. In short, expanded Aspire-T cells were stained with antibodies against CD3, CD8, G4S, CD45RO, and CCR7, and subsequently analyzed by flow cytometry. Different T cell phenotypes included effector memory T cells (“Tem”), central memory T cells (“Tcm”), naive T cells (“Tn”), and terminally differentiated effector T cells (“Teff”). Figure 20A As shown, T cells expressing scFv-ΔCD3e-28z (Design B) exhibited a greater proportion of “Tcm” (i.e., central memory T cells) in culture than T cells expressing scFv-CD3e-28z (Design A). This suggests that T cells expressing scFv-ΔCD3e-28z (Design B) retain more Tcm after expansion compared to T cells expressing scFv-CD3e-28z (Design A). When compared to T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-FOZ-28z (design D) or scFv-ΔCD3e-40DZ-28z (design E) showed a similar memory phenotype, while T cells expressing scFv-ΔCD3eZ-28z (design C) showed a higher proportion of Tcm, and T cells expressing scFv-ΔCD3e-41BBZ-28z (design F) had slightly less, i.e., a lower proportion of Tcm, but more Teff (i.e., effector T cells). Furthermore, as... Figure 20BAs shown, T cells expressing scFv-ΔCD3e-41BBM-M1Z-28z (design G) or scFv-ΔCD3e-CD40M-M1Z-28z (design H) exhibited more Tcm in culture than T cells expressing scFv-ΔCD3e-28z (design B). However, T cells expressing scFv-ΔCD3e-28z-S3 (design I), scFv-ΔCD3e-S5-28z-S3 (design J), and scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed slightly less Tcm but more Teff. In particular, T cells expressing scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed the highest Teff. T cells expressing scFv-ΔCD3e-CD40M-M1Z-28z (design H) showed higher levels of Tn (naive T cells) than scFv-ΔCD3e-28z (design B) or scFv-ΔCD3e-41BBM-M1Z-28z (design G).

[0377] 4.4. In vitro cytotoxicity and activation of Aspire-T cells against target tumor cells

[0378] Further examination was conducted to determine the specific in vitro cytotoxicity against the target tumor cells. In short, different Aspire-T cells were cultured overnight with CFSE-prelabeled target tumor cells (SiHa cells), after which tumor cells were harvested and their viability was measured using 7-AAD. Figure 21A As shown, no cytotoxicity was detected in the control "NT" T cells, and T cells expressing scFv-ΔCD3e-28z (design B), scFv-ΔCD3e-FOZ-28z (design D), scFv-ΔCD3e-40DZ-28z (design E), or scFv-ΔCD3e-41BBZ-28z (design F) showed similar T cell cytotoxicity against target SiHa tumor cells compared to T cells expressing scFv-CD3e-28z (design A). Figure 21BAs shown, compared with T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-41BBM-M1Z-28z (design G), scFv-ΔCD3e-28z-S3 (design I), or scFv-ΔCD3e-S5-28z-S3 (design J) exhibited similar T cell killing activity, while T cells expressing scFv-ΔCD3e-CD40M-M1Z-28z (design H) showed lower T cell killing activity against tumor target cells, and T cells expressing scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed higher T cell killing activity against tumor target cells.

[0379] Furthermore, the activation of Aspire-T cells after antigen-specific stimulation was examined in vitro. In short, different Aspire-T cells were cultured for 48 hours with and without target SiHa tumor cells, after which the supernatant was collected, and IFN-γ levels were measured by ELISA. Figure 22A As shown, compared to T cells expressing scFv-ΔCD3e-28z (Design B), scFv-ΔCD3e-FOZ-28z (Design D), scFv-ΔCD3e-40DZ-28z (Design E), or scFv-ΔCD3e-41BBZ-28z (Design F) exhibited similar T cell activation after antigen stimulation. However, T cells expressing scFv-ΔCD3e-40DZ-28z (Design E) showed higher background activation. T cells expressing scFv-ΔCD3e-41BBZ-28z (Design F) showed higher activation compared to T cells expressing scFv-ΔCD3e-28z (Design B). Figure 22B As shown, compared with T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-41BBM-M1Z-28z (design G), scFv-ΔCD3e-28z-S3 (design I), and scFv-ΔCD3e-S5-28z-S3 (design J) showed similar levels of T cell activation, while T cells expressing scFv-ΔCD3e-CD40M-M1Z-28z (design H) showed lower levels of T cell activation, and T cells expressing scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed higher levels of T cell activation.

[0380] 4.5. Expansion of Aspire-T cells after repeated in vitro antigen stimulation

[0381] In short, different Aspire-T cells are repeatedly stimulated by tumor target cells, and the proliferation of CAR+T cells (i.e., T cells expressing Aspire-TCR) is monitored. For example... Figure 23A As shown, T cells expressing scFv-ΔCD3e-28z (Design B) or scFv-ΔCD3eZ-28z (Design C) exhibited better T cell expansion after repeated antigen stimulation compared to T cells expressing scFv-CD3e-28z (Design A). Figure 23B As shown, compared with T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-41BBM-M1Z-28z (design G), scFv-ΔCD3e-CD40M-M1Z-28z (design H), and scFv-ΔCD3e-S5-28z-S3 (design J) showed similar T cell expansion after repeated antigen stimulation. Compared with T cells expressing scFv-ΔCD3e-28z (design B), T cells expressing scFv-ΔCD3e-28z-S3 (design I) and scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed significantly enhanced T cell expansion after repeated antigen stimulation.

[0382] 4.6. In vivo antitumor efficacy and proliferative capacity of Aspire-T cells armored with membrane-tethered IL12 (mtIL12)

[0383] The in vivo antitumor efficacy and proliferative capacity of Aspire-T cells were further evaluated using an NSG (NOD scid gamma) mouse model. In these characterization experiments, the following constructs were used to transduce T cells to obtain Aspire-T cells: scFv-ΔCD3e-28z (design B), scFv-ΔCD3e-FOZ-28z (design D), scFv-ΔCD3e-CD40M-M1Z-28z (design H), scFv-ΔCD3e-28z-S3 (design I), and scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K). In each of these constructs, the scFv was anti-ALPP A02 scFv (SEQ ID NO:30), and all other sequences (including motif / region / domain sequences and adapter sequences, etc.) were identical to the corresponding constructs described above using anti-ALPP F8 scFv (SEQ ID NO:9). Furthermore, each of the aforementioned Aspire-T cells was further armored (i.e., co-expressed) with membrane-tethered IL12 (i.e., mtIL12), with the structural diagram (IE06) shown in... Figure 24 As shown, the sequence is shown in SEQ ID NO:31.

[0384] In short, 48 female NSG mice were subcutaneously implanted with 5.0 × 10⁻⁶ cells in 100 μl PBS. 6 Target tumor cells. 28 days post-implantation, on day -1, animals were sorted into groups based on tumor size, with each group containing tumors of approximately equal average size of about 58 mm³. Mice were then intravenously injected with 2.5 × 10⁻⁶ tumor cells. 6 CAR+ T cells (i.e., T cells co-expressing each Aspire-TCR construct and mtIL12 IE06 as described above), or an equivalent amount of the indicated non-transduced (“NT”) cells (8.3 × 10⁻⁶). 6 / mouse). Caliper measurements were recorded twice weekly for all animals during the study. Tumor volume was calculated using the following formula: width 2 × length / 2.

[0385] like Figure 25 As shown, in mouse studies, when treated with the same dose of T cells, all armored Aspire-T cells exhibited inhibition of tumor growth compared to control "NT" T cells. More specifically, IE06 armored Aspire-T cells expressing scFv-ΔCD3e-28z-S3 (design I), scFv-ΔCD3e-CD40M-M1Z-28z (design H), or scFv-ΔCD3e-FOZ-28z (design D) showed the highest antitumor activity, followed by IE06 armored Aspire-T cells expressing scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K); in terms of in vivo antitumor efficacy, they all showed higher antitumor activity than IE06 armored Aspire-T cells expressing scFv-ΔCD3e-28z (design B). It should be noted that even IE06-armored Aspire-T cells expressing scFv-ΔCD3e-28z (design B) still showed great anti-tumor efficacy even after about one month.

[0386] In the same mouse experiment, blood samples were collected on specified days (days 3, 11, 19, and 26) after T-cell injection, and the hCD45:mCD45 ratio in peripheral blood was analyzed by FACS. Figure 26As shown, peripheral blood analysis revealed that, compared with IE06-armored Aspire-T cells expressing scFv-ΔCD3e-28z (design B), IE06-armored Aspire-T cells expressing scFv-ΔCD3e-28z (design B) exhibited the highest cell expansion and persistence. Furthermore, IE06-armored Aspire-T cells expressing scFv-ΔCD3e-28z-S3 (design I) and scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed similar T cell expansion.

[0387] Additionally, on day 27 post-T-cell injection, all remaining mice were euthanized, and their tumors were removed from each mouse. The tumors were digested and then passed through a 70 μm filter. The resulting lysates were collected and processed for staining and subsequent FACS analysis. Figure 27 As shown, analysis of tumor-infiltrating T cells revealed that all IE06-armored Aspire-T cells had a higher T cell population in tumor tissue than control "NT" T cells. Furthermore, compared to IE06 armored T cells expressing scFv-ΔCD3e-28z (design B), IE06 armored T cells expressing scFv-ΔCD3e-FOZ-28z (design D) and scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3 (design K) showed similar hCD45:mCD45 ratios in tumor tissues. However, IE06 armored T cells expressing scFv-ΔCD3e-28z-S3 (design I) and scFv-ΔCD3e-CD40M-M1Z-28z (design H) showed higher hCD45:mCD45 ratios than armored T cells expressing scFv-ΔCD3e-28z (design B), with armored T cells expressing scFv-ΔCD3e-CD40M-M1Z-28z (design H) exhibiting the highest hCD45:mCD45 ratio.

[0388] 4.7. Summary of data from the Aspire-TCR construct

[0389] Based on the above data, the following observations are made.

[0390] Design A (scFv-CD3e-28z): It is mainly used as a reference for evaluating other designs.

[0391] Design B (scFv-ΔCD3e-28z): Based on Design A, the ICD (intrinsic domain) of CD3 epsilon (CD3e) was removed. Experimental results showed that it had better cell surface expression than Design A. Surprisingly, in the absence of the intracellular signaling domain, similar cell lysis and T cell activation against the tumor target were observed in Design B, while Design B showed more central memory T cells (Tcm) compared to Design A. After repeated antigen stimulation, Design B showed significantly better T cell expansion than Design A, indicating that Design B is more memory cell-like and has better persistence.

[0392] Design C (scFv-ΔCD3eZ-28z): Building upon Design B, CD3 zeta (CD3z) ITAM 2 & 3 were added to a truncated CD3e to provide stronger downstream signaling (two ITAMs compared to a single ITAM). Surprisingly, experimental results revealed that Design C exhibited very similar T cell activity, in vitro expansion, and T cell proliferation after repeated antigen stimulation compared to Designs B and A, while showing a higher percentage of Tcm, suggesting that CD3z ITAM 2 & 3 can drive a different T cell phenotype than CD3e ITAM.

[0393] Design D (scFv-ΔCD3e-FOZ-28z): First, OX40 has previously been reported to enhance CAR-T cell proliferation and persistence while reducing exhaustion (Sci. Transl. Med. 13, eaba7308 (2021); Int. J. Cancer 129, 2935-2944 (2011)). Second, the use of CD3z ITAM3 instead of its full-length counterpart has been shown to induce preferential differentiation of a long-lived central memory subset in second-generation CD28-based CARs (Nat. Med. 25, 82-88 (2019)). Third, FcεR1γ is a protein encoded by the FCER1G gene. It is a subunit of the high-affinity IgE receptor, which is expressed on mast cells and basophils. There is some evidence that FcεR1γ signaling may play a role in CAR-T cell function. A previous study found that CAR-T cells expressing a chimeric receptor incorporating the FcεR1γ signaling domain exhibited enhanced cytokine production and cytotoxicity against cancer cells (J Transl Med 21, 197 (2023)). Therefore, in Design D, the combination of co-stimulatory signaling of FcεR1γ and OX40, along with the CD3z ITAM3 signaling domain, was added to the CD3e ICD to achieve enhanced and more durable T cell activity. Experimental data revealed that Design D showed similar T cell cytolytic activity and activation after antigen stimulation compared to Design B. Design D also exhibited similar in vitro expansion and memory phenotypes.

[0394] Design E (scFv-ΔCD3e-40DZ-28z): The CD40 intracellular domain can provide co-stimulation to CAR-T cells in a manner different from 4-1BB signaling to activate NF-κB, and subsequently the expression of T cell co-stimulatory molecules (J. Exp. Med. 2003; 198:1023-34). Therefore, co-stimulatory signaling of CD40 and DAP12, as well as the CD3z ITAM3 signaling domain, were added to Design E to achieve enhanced and more durable T cell activity. Experimental data revealed that Design E exhibited similar T cell cytolytic activity and activation after antigen stimulation compared to Design B. It also showed similar memory phenotype analysis, while Design E showed a slower proliferation rate than Design B for in vitro expansion.

[0395] Design F (scFv-ΔCD3e-41BBMZ-28z): 41BB is a key co-stimulatory factor in T cell development and activation, and upon binding to its ligand (CD137L), it enhances T cell survival, proliferation, and cytokine production. T cells expressing anti-CD19 CARs containing CD137 exhibited the greatest anti-leukemic potency and prolonged (>6 months) survival (MolTher. Aug 2009; 17(8):1453-64). In this design, 41BB ICD and CD3z ITAM3 were added to truncated CD3e to provide T cells with additional co-stimulatory signals besides CD28z. Experimental data revealed that Design F exhibited better T cell cytolytic activity and activation after antigen stimulation compared to Design B, which may be related to more Teff and fewer Tcm. For in vitro expansion, Design F showed slightly lower expression and a slower proliferation rate than Design B. Compared to design E, design F showed more Teff, less Tcm, and slightly better cell lysis and T cell activation, which suggests that CD40 co-stimulatory signaling can drive T cells to be more memory-like compared to 4-1BB.

[0396] Design G (scFv-ΔCD3e-41BBM-M1Z-28z): To improve viral transduction efficiency and increase transgene expression, a functional motif was used instead of a domain (e.g., the 4-1BB motif instead of the 4-1BB intracellular domain). Additionally, the linker (LAT) for T cell activation is a pivotal protein that rapidly becomes phosphorylated with tyrosine residues upon TCR pathway activation. This post-translational modification transforms the LAT into a crucial docking platform for proteins containing the SH2 domain. LAT phosphorylation facilitates the assembly of numerous adaptor proteins and signaling molecules into complex multimolecular signaling structures. These complexes can strategically localize near the TCR binding site, thereby coordinating downstream signaling events necessary for T cell activation. In the current design, the LAT motif was added to pre-localize downstream signaling and enhance T cell activity. Experimental data showed that Design G had slightly higher expression than Design B. In memory phenotype analysis, Design G showed significantly more Tcm in culture than Design B, likely due to the synergistic effect of 4-1BB co-stimulation and LAT signaling. Functional validation showed that design G had similar cytolytic activity against tumor targets after repeated antigen stimulation and slightly better amplification, consistent with a higher percentage of Tcm, and indicated better durability.

[0397] Design H (scFv-ΔCD3e-CD40M-M1Z-28z): In this design, unlike Design G, CD40 instead of 4-1BB is used to drive a more stem / memory-like T cell phenotype and better T cell persistence. Similarly, to minimize vector size, the CD40 motif is used instead of the entire ICD. Experimental validation showed that, similar to Design G, Design H had higher viral transduction efficiency and better expression on primary T cells than Design B. It also showed significantly more Tcm and Tn (naive T cells) in culture than Design B, which could explain why Design H had slower proliferation, lower cytolytic activity, and T cell activation against tumor targets. Design H had slightly higher Tn compared to Design G. In in vitro repeated antigen stimulation, Design H showed similar T cell expansion. However, in animal studies, Design H showed significantly better in vivo T cell proliferation and persistence, as well as good antitumor potency.

[0398] In the summary of AH design above, the CD3zeta (CD3z)ITAM motif significantly influences T cell phenotype, thereby promoting a more memory-like state compared to CD3epsilon (CD3e)ITAM; CD40 co-stimulatory signaling can more effectively bias T cells towards a memory-like phenotype than 4-1BB signaling; utilizing smaller motifs can reduce overall vector size, which has been shown to enhance transgene expression in primary T cells; and strategic incorporation of co-stimulatory signaling elements, particularly CD40 or 4-1BB, and LAT, can significantly improve T cell "stemness," leading to improved T cell persistence. These insights pave the way for optimizing T cell-based therapies and could significantly impact the development of more effective and longer-lasting immunotherapies.

[0399] Optimal activation and proliferation of T cells require a triple signaling pathway: TCR conjugation (signal 1), co-stimulation (signal 2), and cytokine conjugation (signal 3). Currently, clinically evaluated CAR constructs contain both a CD3z domain for TCR signaling and a domain for co-stimulation. However, they lack a domain for transmitting the crucial signal 3. Signal 3 is particularly important because it involves cytokines that interact primarily with common γ-chain receptors essential for T cell immunity via the JAK-STAT signaling pathway. Cytokines IL-2, IL-7, and IL-15 primarily activate STAT5 via their associated motif YXXL (SEQ ID NO:23), while IL-21 distinctly activates STAT3 via its associated motif YXXQ (SEQ ID NO:22) within the IL-21 receptor.

[0400] In the following design, a STAT motif is added to the Aspire-TCR construct to enable Aspire-T cells to induce cytokine signaling upon antigen stimulation, thereby achieving better T cell activity.

[0401] Design I (scFv-ΔCD3e-28z-S3): Building upon Design B, the STAT3 binding motif (YRHQ) was incorporated into the expressed CD3z. Compared to Design B, Design I exhibited slightly better Aspire expression in primary T cells and more Teff in culture. Furthermore, better T cell expansion was observed in in vitro repeated antigen stimulation. Consistently, in animal studies, Design I also demonstrated better in vivo proliferation after T cell metastasis, a higher T cell population in tumor tissue, and more significant antitumor activity, suggesting that the addition of STAT3 signaling enhances T cell proliferation and likely also enhances T cell persistence.

[0402] Design J (scFv-ΔCD3e-S5-28z-S3): Based on Design B, which lacks the CD3 epsilon (CD3e) ICD (intracellular domain), CD3 zeta (CD3z) ITAM 2 & 3 were added to the truncated CD3e to potentially provide stronger downstream signaling (two ITAMs compared to a single ITAM). The STAT5 binding motif (YLSL) was incorporated into the CD3z fused to CD3e, and the STAT3 binding motif (YRHQ) was incorporated into the separately expressed CD3z to provide downstream signaling for STAT5 and STAT3 cytokines, aiming for better T cell activity and long-term persistence. Surprisingly, no significantly improved T cell activity similar to Design I was observed in Design J. Experimental results indicate that Design J has similar expression levels compared to Design B. Additionally, in vitro functions (cytotoxicity and T cell activation) differ slightly from Design B. It exhibits similar T cell expansion after repeated antigen stimulation. This may indicate that the combination of CD28 co-stimulation with STAT3 & STAT5 signaling does not provide optimal T cell activation and proliferation.

[0403] Design K (scFv-ΔCD3e-41BBM-M1Z-S5-28z-S3): Based on Design J, the 41BB-LAT motif was incorporated into the design to further improve T cell activation and proliferation. Compared to Design B or Design J, Design K exhibited more Tn and Teff cells. It also demonstrated better T cell expansion with or without repeated antigen stimulation. Functional validation showed that Design K possessed stronger cytolytic activity and higher cytokine production after antigen stimulation. On the other hand, Design K did not show significant antitumor activity, possibly due to its strong IE06 (IL12) armor, which led to more easy exhaustion of Design K cells.

[0404] In summarizing the design of IK, it has been found that integration of cytokine signaling pathways (especially those mediated by STAT3 and / or STAT5) amplifies T cell proliferation, indicating enhanced T cell persistence; and strategic selection of co-stimulatory signals in combination with cytokine signaling pathways is crucial. Specifically, the synergistic effect of 4-1BB (and possibly CD40) with STAT3 and / or STAT5 signaling significantly improves T cell activation and proliferation. This enhancement contributes to improving the potential antitumor efficacy of T cell-based therapies. These findings highlight the importance of fine-tuning T cell therapy to maximize therapeutic outcomes by manipulating both co-stimulation and cytokine signaling.

[0405] Other implementation plans

[0406] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An engineered CD3zeta (CD3z) subunit comprising a co-stimulatory region operatively connected to or incorporated into a CD3zeta component, wherein: The CD3zeta component comprises human CD3zeta or a functional portion or variant thereof, wherein the human CD3zeta or a functional portion or variant thereof comprises a sequence having at least 80% sequence identity with SEQ ID NO:1; and The co-stimulatory region is located within the intracellular domain of the engineered CD3zeta subunit.

2. The engineered CD3zeta subunit according to claim 1, wherein when expressed in immune cells, at least one of the following is satisfied: (1) Immune cells expressing the engineered CD3zeta subunit have reduced activation in the absence of antigen stimulation compared to when immune cells do not express the engineered CD3zeta subunit; (2) Immune cells expressing the engineered CD3zeta subunit have reduced cytotoxicity against non-target cells compared to when immune cells do not express the engineered CD3zeta subunit. (3) Immune cells expressing the engineered CD3zeta subunit exhibit increased activation upon antigen stimulation compared to when immune cells do not express the engineered CD3zeta subunit; and (4) Immune cells expressing the engineered CD3zeta subunit have an increased immune cell response against their corresponding target cells compared to when immune cells do not express the engineered CD3zeta subunit.

3. The engineered CD3zeta subunit according to claim 1 or claim 2, wherein the CD3zeta component comprises the amino acid sequence shown in SEQ ID NO:

1.

4. The engineered CD3zeta subunit according to any one of claims 1-3, wherein the co-stimulatory region comprises a co-stimulatory domain, or a functional portion or functional variant thereof, of a protein selected from the group consisting of CD28, 4-1BB, OX40, CD2, CD27, CDS, ICAM-1, LFA-1, and ICOS.

5. The engineered CD3zeta subunit of claim 4, wherein the co-stimulatory region comprises a co-stimulatory domain of CD28, or a functional portion or variant thereof, and the co-stimulatory domain of CD28, or a functional portion or variant thereof, comprises a sequence having at least 80% sequence identity with SEQ ID NO:

2.

6. The engineered CD3zeta subunit of claim 4, wherein the co-stimulatory region comprises a co-stimulatory domain of 4-1BB, or a functional portion or variant thereof, wherein the co-stimulatory domain of 4-1BB, or a functional portion or variant thereof, comprises a sequence having at least 80% sequence identity with SEQ ID NO:

3.

7. The engineered CD3zeta subunit according to any one of claims 1-6, wherein the co-stimulatory region is operatively fused to the C-terminus of the CD3zeta component.

8. The engineered CD3zeta subunit according to any one of claims 1-6, wherein the co-stimulatory region is located between the transmembrane domain and the intracellular domain of the CD3zeta component.

9. The engineered CD3zeta subunit according to any one of claims 1-8, further comprising a STAT-binding region within its intracellular domain.

10. The engineered CD3zeta subunit of claim 9, wherein the STAT binding region comprises a STAT3 binding motif, the STAT3 binding motif comprising at least 50%, 75%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

22.

11. The engineered CD3zeta subunit of claim 10, wherein the STAT3 binding motif comprises at least 50%, 75%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

21.

12. The engineered CD3zeta subunit of claim 9, wherein the STAT binding region comprises a STAT5 binding motif, the STAT5 binding motif comprising at least 50%, 75%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

23.

13. The engineered CD3zeta subunit of claim 12, wherein the STAT5 binding motif comprises at least 50%, 75%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

24.

14. The engineered CD3zeta subunit according to any one of claims 9-13, wherein the STAT binding region is located in the region of the CD3zeta component corresponding to position 150-164 of SEQ ID NO:

1.

15. The engineered CD3zeta subunit of claim 14, wherein the STAT binding region is inserted at a position between position 157 and position 158 of the CD3zeta component corresponding to SEQ ID NO:

1.

16. The engineered CD3zeta subunit of claim 15, wherein the CD3zeta component comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

20.

17. The engineered CD3zeta subunit of claim 15, wherein the CD3zeta component comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

29.

18. The engineered CD3zeta subunit according to any one of claims 9-17, wherein when the engineered CD3zeta subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the STAT-binding region is not present in the engineered CD3zeta subunit: The T cell population exhibited increased in vitro expansion after repeated stimulation with the antigen; The T cell population exhibits increased potency against tumors containing the target cells in vivo; and The T cell population exhibits a higher proportion in vivo in tumor tissues infiltrated by it.

19. The engineered CD3zeta subunit according to any one of claims 1-18, wherein the immune cell is a T lymphocyte, a tumor-infiltrating lymphocyte (TIL), or a natural killer (NK) cell.

20. The engineered CD3zeta subunit according to any one of claims 1-19, wherein when the engineered CD3zeta subunit is expressed in a population of T cells expressing the T cell receptor (TCR), the T cell population exhibits increased surface expression compared to when the co-stimulatory region is not present in the engineered CD3zeta subunit.

21. A method for regulating the activity of immune cells, comprising: The engineered CD3zeta subunit according to any one of claims 1-20 is expressed in the immune cells.

22. The method of claim 21, wherein the immune cells expressing the engineered CD3 zeta subunit: (1) Reduced activation in the absence of antigen stimulation compared to when immune cells do not express the engineered CD3zeta subunit; (2) It exhibits reduced cytotoxicity against non-target cells compared to when immune cells do not express the engineered CD3zeta subunit; (3) Increased activation upon antigen stimulation compared to when immune cells do not express the engineered CD3zeta subunit; or (4) It has an increased immune cell response against its corresponding target cells compared to when immune cells do not express the engineered CD3zeta subunit.

23. An engineered CD3epsilon (CD3e) subunit comprising a target recognition region and a CD3epsilon component, wherein: The target recognition region is operatively fused to the extracellular domain of the engineered CD3epsilon subunit; and The CD3epsilon component comprises a truncated CD3epsilon, wherein the truncation is located in the region of the human CD3epsilon subunit corresponding to its intercellular domain.

24. The engineered CD3epsilon subunit according to claim 23, wherein the truncation is located in the region corresponding to positions 151-179 of the human CD3epsilon subunit in SEQ ID NO:

5.

25. The engineered CD3epsilon subunit of claim 24, wherein the CD3epsilon component comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

10.

26. The engineered CD3epsilon subunit of claim 25, wherein the CD3epsilon component comprises the amino acid sequence of SEQ ID NO:

10.

27. The engineered CD3epsilon subunit according to any one of claims 23-26, wherein when the engineered CD3epsilon subunit is expressed in a T cell population, the T cell population exhibits increased surface expression and / or increased amplification of the engineered CD3epsilon subunit compared to when the CD3epsilon component comprises a full-length CD3epsilon subunit.

28. The engineered CD3epsilon subunit according to any one of claims 23-26, wherein the C-terminus of the CD3epsilon component is the C-terminus of the engineered CD3epsilon subunit.

29. The engineered CD3epsilon subunit of claim 28, wherein when the engineered CD3epsilon subunit is expressed in a T cell population, at least one of the following is satisfied: (1) The T cell population exhibited increased surface expression of the engineered CD3epsilon subunit compared to when the CD3epsilon component contained a full-length CD3epsilon subunit; (2) The T cell population contains a higher percentage of central memory T cells compared to when the CD3epsilon component contains a full-length CD3epsilon subunit; and (3) The T cell population exhibited increased in vitro expansion after repeated stimulation with the antigen compared to when the CD3epsilon component contained a full-length CD3epsilon subunit.

30. The engineered CD3epsilon subunit according to any one of claims 23-26, further comprising at least one functional region in its intracellular domain, wherein the at least one functional region is operatively fused to the C-terminus of the CD3epsilon component.

31. The engineered CD3epsilon subunit of claim 30, wherein the at least one functional region comprises an immune receptor tyrosine-based activation motif (ITAM).

32. The engineered CD3epsilon subunit of claim 31, wherein the ITM is derived from CD3z, CD3g, DAP12, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD28, or CD72.

33. The engineered CD3epsilon subunit of claim 32, wherein the ITAM comprises: (1) CD3z ITAM3, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:11; or (2) CD3z ITAM2-3, which contains at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:

12.

34. The engineered CD3epsilon subunit according to any one of claims 31-33, wherein the ITAM is located at the C-terminus of the engineered CD3epsilon subunit.

35. The engineered CD3epsilon subunit of claim 34, wherein the ITAM is operatively fused to the C-terminus of the CD3epsilon component, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3.

36. The engineered CD3epsilon subunit of claim 35, wherein the ITAM comprises a STAT binding region, wherein the STAT binding region is a STAT3 binding motif or a STAT5 binding motif.

37. The engineered CD3epsilon subunit according to any one of claims 31-36, wherein when the engineered CD3epsilon subunit is expressed in a T cell population, the T cell population contains a higher percentage of central memory T cells compared to when the ITAM is not present in the engineered CD3epsilon subunit.

38. The engineered CD3epsilon subunit according to any one of claims 30-37, wherein the at least one functional region comprises any one or a combination of the following: (1) FcεR1γ intracellular domain containing at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:25; (2) OX40 intracellular domain, which contains at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:26; (3) CD40 intracellular domain, which contains at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:27; (4) DAP12 intracellular domain containing at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:28; (5) 4-1BB intracellular domain containing at least 80%, 85%, 90%, 95%, or 100% of the amino acid sequence identical to that of SEQ ID NO:3; (6) 4-1BB motif, which contains at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:15; (7) CD40 motif, which contains at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:17; (8) A linker (LAT) motif for activating T cells, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:16; and (9) CD28 intracellular domain containing at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:

2.

39. The engineered CD3epsilon subunit according to any one of claims 23-38, further comprising a STAT-binding region within its intracellular domain.

40. The engineered CD3epsilon subunit of claim 39, wherein the STAT binding region comprises: (1) A STAT3 binding motif comprising an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of SEQ ID NO:22; or (2) STAT5 binding motif, which contains at least 50%, 75%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

23.

41. The engineered CD3epsilon subunit of claim 40, wherein the STAT binding region comprises a STAT3 binding motif, the STAT3 binding motif comprising an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of SEQ ID NO:

21.

42. The engineered CD3epsilon subunit of claim 40, wherein the STAT binding region comprises a STAT5 binding motif, the STAT5 binding motif comprising an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of SEQ ID NO:

24.

43. The engineered CD3epsilon subunit according to any one of claims 39-42, further comprising ITAM in its intracellular domain, wherein ITAM comprises the STAT binding region.

44. The engineered CD3epsilon subunit of claim 43, wherein the ITAM is a CD3zITAM2-3 motif, the CD3zITAM2-3 motif comprising at least 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:

18.

45. The engineered CD3epsilon subunit of claim 43, wherein the ITAM is a CD3z ITAM3 motif, the CD3z ITAM3 motif comprising at least 80%, 85%, 90%, 95%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:

19.

46. ​​The engineered CD3epsilon subunit according to claim 38, wherein the at least one functional region comprises both the FcεR1γ intracellular domain and the OX40 intracellular domain.

47. The engineered CD3epsilon subunit of claim 46, wherein the at least one functional region comprises a first complex functional region, the first complex functional region comprising the FcεR1γ intracellular domain and the OX40 intracellular domain in the direction from the N-terminus to the C-terminus, wherein the first complex functional region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

13.

48. The engineered CD3epsilon subunit according to claim 46 or 47, wherein the at least one functional region further comprises ITAM at the C-terminus of the first composite functional region, wherein the ITAM comprises CD3zITAM3 or CD3zITAM2-3.

49. The engineered CD3zeta subunit of claim 48, wherein when the engineered CD3zeta subunit is expressed in a T cell population, the T cell population exhibits increased potency against tumors containing target cells in vivo compared to when the FcεR1γ intracellular domain, OX40 intracellular domain, and ITAM are absent in the engineered CD3zeta subunit.

50. The engineered CD3epsilon subunit of claim 38, wherein the at least one functional region comprises both the CD40 intracellular domain and the DAP12 intracellular domain.

51. The engineered CD3epsilon subunit of claim 50, wherein the at least one functional region comprises a second complex functional region, the second complex functional region comprising the CD40 intracellular domain and the DAP12 intracellular domain in the direction from the N-terminus to the C-terminus, wherein the second complex functional region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

14.

52. The engineered CD3epsilon subunit according to claim 50 or claim 51, wherein the at least one functional region further comprises ITAM at the C-terminus of the second composite functional region, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3.

53. The engineered CD3epsilon subunit according to any one of claims 23-52, further comprising a 4-1BB intracellular domain and an ITAM from its N-terminus to its C-terminus, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3.

54. The engineered CD3epsilon subunit of claim 53, wherein when the engineered CD3epsilon subunit is expressed in T cells specifically targeting cells with a specific antigen, it satisfies at least one of the following conditions compared to when the 4-1BB intracellular domain and ITAM are not present in the engineered CD3epsilon subunit: The T cells exhibited increased cytotoxicity against the target cells in vitro; and The T cells exhibited increased stimulation by the antigen in vitro.

55. The engineered CD3epsilon subunit of claim 38, wherein the at least one functional region comprises both a 4-1BB motif and a LAT motif.

56. The engineered CD3epsilon subunit of claim 55, further comprising ITAM at the C-terminus of the engineered CD3epsilon subunit, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3.

57. The engineered CD3epsilon subunit of claim 56, wherein when the engineered CD3epsilon subunit is expressed in a T cell population, it satisfies at least one of the following conditions compared to when the 4-1BB motif, LAT motif, and ITAM are all absent in the engineered CD3epsilon subunit: The T cell population contains a high percentage of central memory T cells; and The T cell population exhibited increased in vitro expansion after repeated stimulation with the antigen.

58. The engineered CD3epsilon subunit of claim 56, wherein the ITAM comprises a STAT binding region, wherein the STAT binding region is a STAT3 binding motif or a STAT5 binding motif.

59. The engineered CD3epsilon subunit according to claim 38, wherein the at least one functional region comprises both the CD40 motif and the LAT motif.

60. The engineered CD3epsilon subunit of claim 59, further comprising ITAM at the C-terminus of the engineered CD3epsilon subunit, wherein the ITAM comprises CD3z ITAM3 or CD3z ITAM2-3.

61. The engineered CD3zeta subunit of claim 60, wherein when the engineered CD3zeta subunit is expressed in a T cell population, it satisfies at least one of the following conditions compared to when the CD40 motif, LAT motif, and ITAM are all absent in the engineered CD3zeta subunit: The T cell population exhibited increased surface expression of the engineered CD3epsilon subunit; The T cell population contains a high percentage of naive T cells; The T cell population contains a high percentage of central memory T cells; The T cell population exhibits increased potency against tumors containing target cells in vivo; and The T cell population exhibits a higher proportion in vivo in tumor tissues infiltrated by it.

62. The engineered CD3epsilon subunit according to any one of claims 23-61, wherein the target recognition region is operatively incorporated into the CD3epsilon component.

63. The engineered CD3epsilon subunit according to any one of claims 23-61, wherein the target recognition region comprises: (1) Antigen-binding region; or (2) Ligands or fragments thereof that bind to cell surface receptors expressed on target cells of immune cells.

64. The engineered CD3epsilon subunit of claim 63, wherein the antigen-binding region comprises a single-stranded variable fragment (scFv) that specifically recognizes ALPP, LYPD3, IL13Ra2, BCMA, CD16, CD19, CD20, CD22, CD27, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, mesothelin, NKG2D, phosphatidylinositol proteoglycan-3 (GPC-3), FAP, FRa (folate receptor α), EGFR, EGFR vIII, IL-11Ra (IL11 receptor α), VEGFR-II, B7-H6, and DNAM-1.

65. The engineered CD3epsilon subunit of claim 64, wherein the antigen-binding region comprises a single-stranded variable fragment (scFv) targeting ALPP.

66. The engineered CD3epsilon subunit of claim 63, wherein the antigen-binding region comprises a single-domain antibody (sdAb or nanobody).

67. The engineered CD3epsilon subunit of claim 63, wherein the ligand is selected from the group consisting of: IL13 (E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRINB2, CTLX, LFA-1, and FSH.

68. The engineered CD3epsilon subunit according to claim 67, wherein the ligand is IL13 (E13Y).

69. The engineered CD3epsilon subunit according to any one of claims 23-68, further comprising a flexible joint between the target recognition region and the CD3epsilon component.

70. The engineered CD3epsilon subunit of claim 69, wherein the flexible connector comprises the sequence of SEQ ID NO:

6.

71. An engineered TCR complex system comprising at least one of the following: The engineered CD3zeta subunit according to any one of claims 1-20; and The engineered CD3epsilon subunit according to any one of claims 23-70.

72. The engineered TCR complex system of claim 71, comprising both the engineered CD3epsilon subunit and the engineered CD3zeta subunit.

73. The engineered TCR complex system of claim 72, wherein the co-stimulatory region of the engineered CD3zeta subunit comprises: The co-stimulatory domain of CD28, or its functional portion or functional variant; or The costimulatory domain of 4-1BB, or its functional portion or functional variant.

74. The engineered TCR complex system of claim 72 or 73, wherein one or both of the engineered CD3epsilon subunit and the engineered CD3zeta subunit contain a STAT-binding region within their intracellular domain.

75. The engineered TCR complex system of claim 74, wherein both the engineered CD3epsilon subunit and the engineered CD3zeta subunit contain a STAT-binding region within their intracellular domains, wherein the STAT-binding region of the engineered CD3epsilon subunit is different from the STAT-binding region of the engineered CD3zeta subunit.

76. The engineered TCR complex system according to claim 75, wherein the STAT binding region of the engineered CD3epsilon subunit and the STAT binding region of the engineered CD3zeta subunit (1) are respectively a STAT3 binding motif and a STAT5 binding motif, or (2) are respectively a STAT5 binding motif and a STAT3 binding motif.

77. The engineered TCR complex system of claim 74, wherein only the engineered CD3zeta subunit contains a STAT-binding region within its intracellular domain, wherein the STAT-binding region contains a STAT3-binding motif or a STAT5-binding motif.

78. The engineered TCR complex system according to any one of claims 74-77, wherein when the engineered CD3epsilon subunit is expressed in a T cell population, at least one of the following is satisfied compared to when the STAT binding region is not present in either the engineered CD3epsilon subunit or the engineered CD3zeta subunit: The T cell population contains a high percentage of terminally differentiated effector T cells; The T cell population exhibited increased antigen-specific stimulation in vitro; and The T cell population exhibited increased in vitro expansion after repeated stimulation with the antigen.

79. The engineered TCR complex system of claim 71, comprising the engineered CD3zeta subunit, and further comprising an engineered target-recognition TCR subunit, wherein the TCR subunit is based on one of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, or CD3δ, and preferably based on one of TCRα, TCRβ, CD3ε, CD3γ, or CD3δ.

80. The engineered TCR complex system of claim 79, wherein the engineered target-recognition TCR subunit includes a target-recognition region, the target-recognition region comprising: (1) An antigen-binding region comprising at least one of a single-chain variable fragment (scFv) or a single-domain antibody; or (2) Ligands or fragments thereof that bind to cell surface receptors expressed on target cells of immune cells.

81. A chimeric polypeptide comprising a target recognition region operatively linked to or incorporated into one of the TCR alpha subunit, TCR beta subunit, CD3gamma subunit, CD3delta subunit, or CD3epsilon subunit, or a functional portion or functional variant thereof, wherein: The target recognition region includes a ligand, a functional portion thereof, or a functional variant thereof, wherein when the chimeric polypeptide is expressed in immune cells, the target recognition region of the chimeric polypeptide is capable of binding to a cell surface receptor expressed on a target cell of the immune cell.

82. The chimeric polypeptide of claim 81, wherein the target recognition region is operatively linked to or incorporated into a CD3epsilon subunit or a functional portion or variant thereof.

83. The chimeric polypeptide of claim 82, wherein the CD3epsilon subunit comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

5.

84. The chimeric polypeptide of claim 82, wherein the CD3epsilon subunit comprises a truncated CD3epsilon, the truncated CD3epsilon comprising at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

10.

85. The chimeric polypeptide according to any one of claims 81-84, wherein the ligand is selected from the group consisting of: IL13 (E13Y), IL-13, IL-11, IL-10, APRIL, GM-CSF, TPO, Adnectin, T1E, FLT3L, EPHRINB2, CTLX, LFA-1, and FSH.

86. The chimeric polypeptide according to claim 85, wherein the ligand is IL13 (E13Y).

87. The chimeric polypeptide of claim 86, wherein the IL13(E13Y) ligand comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:

4.

88. An engineered immune cell comprising: The engineered TCR complex system according to any one of claims 71-80; and / or The chimeric polypeptide according to any one of claims 79-85.

89. The engineered immune cell according to claim 88, wherein the engineered immune cell is a T lymphocyte, a tumor-infiltrating lymphocyte (TIL), or a natural killer (NK) cell.

90. A method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of engineered immune cells according to claim 88 or claim 89.

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