Chimeric polypeptides for regulating immune cells

Abstract

The present invention relates to chimeric polypeptides, nucleic acids encoding such chimeric polypeptides, cells, preferably T cells, CAR T cells, NK cells, or CAR NK cells, comprising the chimeric polypeptides of such nucleic acids, and methods for treating cancer, particularly immune cell therapy, using such cells and polypeptides of nucleic acids.The chimeric polypeptides of the present invention can reversibly and dose-dependently regulate the functions (cytokine release, cytotoxicity) of T cells and NK cells.

Description

[Technical Field]

[0001] The present invention relates to chemically controlled chimeric polypeptides that can reversibly and dose-dependently control the function of T cell receptors, NK cell receptors, and / or chimeric antigen receptors. [Background technology]

[0002] Over the past few years, numerous adoptive T-cell therapies have been developed for the treatment of both hematological and solid tumors (Rosenberg et al, Science. 2015 Apr 3;348(6230):62-8, June et al, Science. 2018 Mar 23;359(6382):1361-1365). Specifically, following the discovery of the antitumor effect of donor-derived T cells associated with allogeneic hematopoietic stem cell transplantation, donor lymphocyte infusion (DLI) has been developed as a method to utilize this effect (Frey et al, Best Pract Res Clin Haematol. 2008 Jun; 21(2): 205-222).

[0003] Recently, T cells modified with chimeric antigen receptors (CARs) or T cell receptors (TCRs) have been used to provide patients with tumor-responsive T cell populations, and the remarkable clinical activity of CD19 CAR T cells in patients with B-cell malignancies has led to the recent approval of these T cell products for B-ALL and DLBCL (Boyiadzis et al, J Immunother Cancer. 2018 Dec 4;6(1):137).

[0004] Importantly, the broader application of adoptive T-cell therapy in cancer is currently limited by safety concerns. Specifically, when antigens targeted by the adoptive T-cell pool are expressed in healthy tissue, on-target-off tumor toxicity is often observed (Bonifant et al, Mol Ther Oncolytics. 2016 Apr 20;3:16011).

[0005] In recent years, considerable effort has been made to discover truly cancer-specific markers in order to avoid damage to normal cells. However, it has been found that cancer-specific markers expressed in tumors of many patients are extremely rare. Furthermore, unexpected cross-reactivity between autoantigens expressed at low levels in important tissues and recombinant T cells is associated with severe toxicity (Morgan et al, J Immunother. 2013 Feb;36(2):133-51).

[0006] Finally, even if complete tumor-specific activation of injected cells can be achieved, the severe cytokine release upon T cell recognition of large tumor masses raises safety concerns, so any method to control the degree of T cell activation would be very appealing.

[0007] Due to the high risk of treatment-induced toxicity in adoptive T-cell therapy, many research groups have developed genetically encoded suicide switches that can be activated by drug administration the moment significant toxicity is observed, such as cell surface marker suicide switches based on HSV-TK, iCas9, and CD20 (Jones et al, Front Pharmacol. 2014 Nov 27;5:254).

[0008] Such suicide switches have primarily been used to configure DLIs, where graft-versus-tumor (GvT) effects correlate with graft-versus-host disease (GvHD), and a balance between these two effects is required. However, the dual nature of these switches inhibits the modulation of T cell function, and these systems have found limited applications in the context of TCR / CAR-modified T cells.

[0009] More recent studies have engineered safety switch technologies that can reversibly control chimeric antigen receptor (CAR) T cells (Wu et al, Science. 2015 Oct 16;350(6258):aab4077, Ma et al, Proc Natl Acad Sci US A. 2016 Jan 26;113(4):E450-8, Loureiro et al, Blood Cancer J. 2018 Sep; 8(9): 81), but these systems cannot be used to regulate the activity of either TCR-modified or unmodified T cells.

[0010] Furthermore, acute GvHD has been observed in clinical trials using T-cell-deficient NK cell therapy, suggesting that a safety switch to control NK cell activity is also desirable (Shah et al, Blood. 2015 Jan 29;125(5):784-92). [Overview of the Initiative]

[0011] From this perspective, products, compositions, methods, and applications that control the function of TCR and CAR T cells and natural killer (NK) cells (i.e., the function of NK cell receptors) in patients are highly desirable, but are not yet readily available. In particular, there is a clear need for reliable, efficient, and reproducible products, compositions, methods, and applications that control the function of TCR / CAR T cells and NK cells, such as the cytotoxic activity of T cells containing such TCRs or CARs, the function of NK cells containing NK cell receptors (NKRs) or CARs, and / or control cytokine secretion by such T cells or NK cells. Therefore, the underlying technical problem of the present invention is to provide such products, compositions, methods, and applications to satisfy any of the aforementioned needs. This technical problem is solved by the claims and embodiments characterized below herein. [Brief explanation of the drawing]

[0012] Embodiments of the present invention will be further described below with reference to the attached drawings.

[0013] [Figure 1-1] Figure 1 shows efficient suppression of T cell function by Zap70's SH2-mediated recruitment of the PD1 tail. Figure 1A is a schematic diagram of the early stages of the TCR signaling pathway, showing the recruitment of Zap70 to the CD3 ζ chain via peptide MHC and PD1-mediated suppression of TCR signaling by PD-L1 ligation. Figure 1B is a schematic diagram of the recruitment of Zap70's SH2-mediated PD1 tail to the activated TCR complex and the resulting inhibition of TCR signaling. Figures 1C and 1D show the results of co-culturing primary human T cells modified with HLA class I restriction CDK4 TCR and Zap70-PD1, Zap70 2xSH2 domain, PD1 tail, or vector control with CDK4 peptide-loaded T2 cells. The data in Figures 1C and 1D show IFNγ, IL2, TNFα production and cell surface LAMP1 expression in CDK4 TCR+EGFP high+CD8 (Figure 1C)+ T cells and +CD4 (Figure 1D)+ T cells.

[0014] [Figure 1-2] Figure 1 shows efficient suppression of T cell function by Zap70's SH2-mediated recruitment of the PD1 tail. Figure 1A is a schematic diagram of the early stages of the TCR signaling pathway, showing the recruitment of Zap70 to the CD3 ζ chain via peptide MHC and PD1-mediated suppression of TCR signaling by PD-L1 ligation. Figure 1B is a schematic diagram of the recruitment of Zap70's SH2-mediated PD1 tail to the activated TCR complex and the resulting inhibition of TCR signaling. Figures 1C and 1D show the results of co-culturing primary human T cells modified with HLA class I restriction CDK4 TCR and Zap70-PD1, Zap70 2xSH2 domain, PD1 tail, or vector control with CDK4 peptide-loaded T2 cells. The data in Figures 1C and 1D show IFNγ, IL2, TNFα production and cell surface LAMP1 expression in CDK4 TCR+EGFP high+CD8 (Figure 1C)+ T cells and +CD4 (Figure 1D)+ T cells.

[0015] [Figure 2-1]Figure 2 is a schematic diagram of the Zap70-PD1-SMASh fusion protein in the absence (Figure 2A) or presence (Figure 2B) of asunaprevir, showing the control of T cell function. In the absence of asunaprevir, the HCV protease releases the Zap70-PD1 portion, inhibiting TCR signaling. In the presence of asunaprevir, the release is blocked, the fusion protein becomes a target of the proteasome, and the inhibition of T cell function is relieved. Figures 2C and 2D show the effect of 24-hour pre-exposure to asunaprevir on the HA signal in intracellular HA staining of primary human T cells modified with the N-terminal HA-tagged Zap70-PD1-SMASh switch or vector control, for EGFP-high-expressing CD8 (Figure 2C) + T cells and CD4 (Figure 2D) + T cells. In Figures 2E - 2H, primary human T cells modified with either the HLA class I-restricted CDK4 TCR and Zap70-PD1-SMASh switch (Figures 2E, 2G) or vector control (Figures 2F, 2H) were pretreated with 10 μM asunaprevir or DMSO control. The data show the intracellular IFNγ, IL2, TNFα and cell surface LAMP1 expression of CDK4 TCR+ EGFP-high +CD8 (Figures 2E - F) + T cells and +CD4 (Figures 2G - H) + T cells when co-cultured with CDK4 peptide-loaded T2 cells in the presence or absence of asunaprevir. Error bars represent the standard deviation (n = 3). The data are representative of two independent experiments.

[0016] [Figure 2-2]Figure 2 is a schematic diagram of the Zap70-PD1-SMASh fusion protein in the absence (Figure 2A) or presence (Figure 2B) of asunaprevir, illustrating the regulation of T cell function. In the absence of asunaprevir, the HCV protease releases the Zap70-PD1 portion, inhibiting TCR signaling. In the presence of asunaprevir, release is blocked, the fusion protein becomes a target of the proteasome, and the inhibition of T cell function is relieved. Figures 2C and 2D show the effect of 24-hour prior exposure to asunaprevir on HA signaling in primary human T cells modified with an N-terminal HA-tagged Zap70-PD1-SMASh switch or vector control, in intracellular HA staining. Figures 2E–2H show primary human T cells modified with either an HLA class I restriction CDK4 TCR and a Zap70-PD1-SMASh switch (Figures 2E, 2G) or a vector control (Figures 2F, 2H), pretreated with 10 μM asunaprevir or DMSO control. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ EGFP-high+CD8 (Figures 2E–F)+ T cells and +CD4 (Figures 2G–H)+ T cells co-cultured with CDK4 peptide-loaded T2 cells in or without asunaprevir. Error bars represent standard deviation (n=3). Data are representative of two independent experiments.

[0017] [Figure 2-3]Figure 2 is a schematic diagram of the Zap70-PD1-SMASh fusion protein in the absence (Figure 2A) or presence (Figure 2B) of asnaperibul, showing the control of T cell function. In the absence of asnaperibul, the HCV protease releases the Zap70-PD1 moiety, inhibiting TCR signaling. In the presence of asnaperibul, release is blocked and the fusion protein becomes a target for the proteasome, relieving the inhibition of T cell function. Figures 2C and 2D are intracellular HA staining of primary human T cells modified with the N-terminal HA-tagged Zap70-PD1-SMASh switch or vector control, showing the effect of 24-hour pre-exposure to asnaperibul on the HA signal of EGFP-high-expressing CD8 (Figure 2C)+ T cells and CD4 (Figure 2D)+ T cells. In Figures 2E - 2H, primary human T cells modified with either the HLA class I-restricted CDK4 TCR and Zap70-PD1-SMASh switch (Figures 2E, 2G) or vector control (Figures 2F, 2H) were pretreated with 10 μM asnaperibul or DMSO control. The data show the intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+ EGFP-high+ CD8 (Figures 2E - F)+ T cells and +CD4 (Figures 2G - H)+ T cells when co-cultured with CDK4 peptide-loaded T2 cells in the presence or absence of asnaperibul. Error bars represent standard deviation (n = 3). The data are representative of two independent experiments.

[0018] [Figure 3-1]Figure 3 shows the bidirectional control of T cell function with adjustable volume, and Figure 3A is a schematic diagram of the experimental procedure. Primary human T cells modified with either an HLA class I restriction CDK4 TCR and Zap70-PD1 or a vector control were pretreated with asunaprevir at the concentrations shown in Figures 3B-D for 24 hours, and then used directly (Figures 3B-C) or cultured in the absence of the drug for 72 hours (Figure 3D). Figures 3B-C show the effect of asunaprevir treatment on the functional output of CD8 (Figure 3B)+ T cells and CD4 (Figure 3C)+ T cells when co-cultured with T2 cells loaded with 10 nM CDK4 peptide in the continuous presence or absence of the illustrated asunaprevir concentrations. Figure 3D shows T cells pretreated with 2.5 μM asunaprevir or DMSO control, cultured for 72 hours in the absence of the drug, and then treated with 2.5 μM asunaprevir or DMSO control, or continued co-culture with 10 nM CDK4 peptide-carrying T2 cells in or without the drug. It should be noted that the initial treatment with asunaprevir did not interfere with subsequent inhibition of T cell function by switching in the absence of the drug (comparing + / - and - / -), and the release of inhibition by asunaprevir was not affected by the release of prior inhibition (comparing - / + and + / +). The data in Figures 3B-D depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0019] [Figure 3-2]Figure 3 shows the bidirectional control of T cell function with adjustable volume, and Figure 3A is a schematic diagram of the experimental procedure. Primary human T cells modified with either an HLA class I restriction CDK4 TCR and Zap70-PD1 or a vector control were pretreated with asunaprevir at the concentrations shown in Figures 3B-D for 24 hours, and then used directly (Figures 3B-C) or cultured in the absence of the drug for 72 hours (Figure 3D). Figures 3B-C show the effect of asunaprevir treatment on the functional output of CD8 (Figure 3B)+ T cells and CD4 (Figure 3C)+ T cells when co-cultured with T2 cells loaded with 10 nM CDK4 peptide in the continuous presence or absence of the illustrated asunaprevir concentrations. Figure 3D shows T cells pretreated with 2.5 μM asunaprevir or DMSO control, cultured for 72 hours in the absence of the drug, and then treated with 2.5 μM asunaprevir or DMSO control, or continued co-culture with 10 nM CDK4 peptide-carrying T2 cells in or without the drug. It should be noted that the initial treatment with asunaprevir did not interfere with subsequent inhibition of T cell function by switching in the absence of the drug (comparing + / - and - / -), and the release of inhibition by asunaprevir was not affected by the release of prior inhibition (comparing - / + and + / +). The data in Figures 3B-D depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0020] [Figure 3-3]Figure 3 shows the bidirectional control of T cell function with adjustable volume, and Figure 3A is a schematic diagram of the experimental procedure. Primary human T cells modified with either an HLA class I restriction CDK4 TCR and Zap70-PD1 or a vector control were pretreated with asunaprevir at the concentrations shown in Figures 3B-D for 24 hours, and then used directly (Figures 3B-C) or cultured in the absence of the drug for 72 hours (Figure 3D). Figures 3B-C show the effect of asunaprevir treatment on the functional output of CD8 (Figure 3B)+ T cells and CD4 (Figure 3C)+ T cells when co-cultured with T2 cells loaded with 10 nM CDK4 peptide in the continuous presence or absence of the illustrated asunaprevir concentrations. Figure 3D shows T cells pretreated with 2.5 μM asunaprevir or DMSO control, cultured for 72 hours in the absence of the drug, and then treated with 2.5 μM asunaprevir or DMSO control, or continued co-culture with 10 nM CDK4 peptide-carrying T2 cells in or without the drug. It should be noted that the initial treatment with asunaprevir did not interfere with subsequent inhibition of T cell function by switching in the absence of the drug (comparing + / - and - / -), and the release of inhibition by asunaprevir was not affected by the release of prior inhibition (comparing - / + and + / +). The data in Figures 3B-D depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0021] [Figure 4-1]Figure 4 shows a schematic diagram of the Zap70-PD1-FKBP12F36V fusion protein in the absence (Figure 4A) or presence (Figure 4B) of the dTAG-13 PROTAC, in the regulation of T cell activity in the CRASH-IT platform using PROTAC. Figures 4C-E show primary human T cells modified with CDK4 TCR, Zap70-PD1-SMASh, Zap70-PD1-FKBP12F36V, or a vector control, pretreated with specified concentrations of the HCV NS3 / 4A protease inhibitor asunaprevir (Figure 4C), grazoprevir (Figure 4D), or dTAG-13 (Figure 4E). The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CD8+ CDK4 TCR+ EGFP-high T cells co-cultured with NKIRTIL006 tumor cells expressing the CDK4 epitope, under continuous administration of indicated concentrations of asunaprevir, grazoprevir, or dTAG-13. Figures 4F-4G show the regulation of T cell cytotoxicity by a small molecule-inducible Zap70-PD1-FKBP12F36V switch. Primary human T cells modified with either an HLA class I-restricted CDK4 TCR and either Zap70-PD1-FKBP12F36V (Figure 4F) or a vector control (Figure 4G) were sorted for CD8+ and high EGFP expression, expanded by REP, and pretreated with 0.5 μM dTAG-13 or DMSO. The data depict 51Cr release from labeled NKIRTIL006 tumor cells when co-cultured with selected CD8+ CDK4 TCR+ EGFP hyper-T cells in the presence or absence of dTAG-13. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0022] [Figure 4-2]Figure 4 shows a schematic diagram of the Zap70-PD1-FKBP12F36V fusion protein in the absence (Figure 4A) or presence (Figure 4B) of the dTAG-13 PROTAC, in the regulation of T cell activity in the CRASH-IT platform using PROTAC. Figures 4C-E show primary human T cells modified with CDK4 TCR, Zap70-PD1-SMASh, Zap70-PD1-FKBP12F36V, or a vector control, pretreated with specified concentrations of the HCV NS3 / 4A protease inhibitor asunaprevir (Figure 4C), grazoprevir (Figure 4D), or dTAG-13 (Figure 4E). The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CD8+ CDK4 TCR+ EGFP-high T cells co-cultured with NKIRTIL006 tumor cells expressing the CDK4 epitope, under continuous administration of indicated concentrations of asunaprevir, grazoprevir, or dTAG-13. Figures 4F-4G show the regulation of T cell cytotoxicity by a small molecule-inducible Zap70-PD1-FKBP12F36V switch. Primary human T cells modified with either an HLA class I-restricted CDK4 TCR and either Zap70-PD1-FKBP12F36V (Figure 4F) or a vector control (Figure 4G) were sorted for CD8+ and high EGFP expression, expanded by REP, and pretreated with 0.5 μM dTAG-13 or DMSO. The data depict 51Cr release from labeled NKIRTIL006 tumor cells when co-cultured with selected CD8+ CDK4 TCR+ EGFP hyper-T cells in the presence or absence of dTAG-13. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0023] [Figure 4-3]Figure 4 shows a schematic diagram of the Zap70-PD1-FKBP12F36V fusion protein in the absence (Figure 4A) or presence (Figure 4B) of the dTAG-13 PROTAC, in the regulation of T cell activity in the CRASH-IT platform using PROTAC. Figures 4C-E show primary human T cells modified with CDK4 TCR, Zap70-PD1-SMASh, Zap70-PD1-FKBP12F36V, or a vector control, pretreated with specified concentrations of the HCV NS3 / 4A protease inhibitor asunaprevir (Figure 4C), grazoprevir (Figure 4D), or dTAG-13 (Figure 4E). The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CD8+ CDK4 TCR+ EGFP-high T cells co-cultured with NKIRTIL006 tumor cells expressing the CDK4 epitope, under continuous administration of indicated concentrations of asunaprevir, grazoprevir, or dTAG-13. Figures 4F-4G show the regulation of T cell cytotoxicity by a small molecule-inducible Zap70-PD1-FKBP12F36V switch. Primary human T cells modified with either an HLA class I-restricted CDK4 TCR and either Zap70-PD1-FKBP12F36V (Figure 4F) or a vector control (Figure 4G) were sorted for CD8+ and high EGFP expression, expanded by REP, and pretreated with 0.5 μM dTAG-13 or DMSO. The data depict 51Cr release from labeled NKIRTIL006 tumor cells when co-cultured with selected CD8+ CDK4 TCR+ EGFP hyper-T cells in the presence or absence of dTAG-13. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0024] [Figure 5-1]Figure 5 shows the functional regulation of CAR-T cells by CRASH-IT, and Figure 5A is a schematic diagram of a second-generation anti-CD19-CD28-CD3ζ CAR interacting with the Zap70-PD1-SMASh switch. Figure 5B shows CD19 expression in K562, Daudi, and Raji tumors. Cells were stained with anti-CD19-PE (solid line) or isotype control PE (dashed line). In Figures 5C-F, primary human T cells modified with either anti-CD19-CD28-CD3ζ CAR and Zap70-PD1-SMASh (Figures 5C, 5E), or vector control (Figures 5D, 5F) were pretreated with 10 μM asunaprevir or DMSO control. The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CAR+ EGFP-high CD8+ (Figure 5C-D)+ T cells and CD4+ (Figure 5E-F)+ T cells co-cultured with CD19-negative K562 or CD19-positive Daudi or Raji tumor cells in or without asunaprevir. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0025] [Figure 5-2]Figure 5 shows the functional regulation of CAR-T cells by CRASH-IT, and Figure 5A is a schematic diagram of a second-generation anti-CD19-CD28-CD3ζ CAR interacting with the Zap70-PD1-SMASh switch. Figure 5B shows CD19 expression in K562, Daudi, and Raji tumors. Cells were stained with anti-CD19-PE (solid line) or isotype control PE (dashed line). In Figures 5C-F, primary human T cells modified with either anti-CD19-CD28-CD3ζ CAR and Zap70-PD1-SMASh (Figures 5C, 5E), or vector control (Figures 5D, 5F) were pretreated with 10 μM asunaprevir or DMSO control. The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CAR+ EGFP-high CD8+ (Figure 5C-D)+ T cells and CD4+ (Figure 5E-F)+ T cells co-cultured with CD19-negative K562 or CD19-positive Daudi or Raji tumor cells in or without asunaprevir. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0026] [Figure 6] Figure 6 shows the regulation of NY-ESO-1 TCR T cell function by CRASH-IT. Figures 6A-D are graphs of primary human T cells modified with either an intermediate affinity HLA class I restriction NY-ESO-1 TCR and a Zap70-PD1-SMASh switch (Figures 6A, 6C), or a vector control (Figures 6B, 6D), pretreated with 10 μM asunaprevir or DMSO control. The data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in NY-ESO-1 TCR+ EGFP high CD8+ (Figures 6A-B)+ T cells and CD4+ (Figures 6C-D)+ T cells in co-culture with NY-ESO-1 peptide-loaded T2 cells in the continuous presence or absence of asunaprevir. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0027] [Figure 7-1]Figure 7 shows the adjustment of the repressive switch by N-terminal modification. Figures 7A-B are graphs of primary human T cells modified with HLA class I restriction CDK4 TCR and Zap70-PD1-SMASh, N-terminal alanine-added (modified) tuZap70-PD1-SMASh, or vector control, pretreated with 10 μM asunaprevir or DMSO control. The data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ EGFP high CD8+ (Figure 7A)+ T cells and CD4+ (Figure 7B)+ T cells in co-culture with NKIRTIL006 tumor cells in the continuous presence or absence of asunaprevir. Notably, the tuZap70-PD1-SMASh switch results in nearly equivalent inhibition of CD4+ T cell function in the absence of asunaprevir, while significantly increasing the recovery of T cell function in the presence of asunaprevir. Figures 7C–D are graphs of primary human T cells modified with either HLA class II restriction CMV TCR and tuZap70-PD1-SMASh (Figure 7C) or a vector control (Figure 7D), pretreated with 10 μM asunaprevir or DMSO control. The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CD4+ CMV TCR+ EGFP hyper-T cells co-cultured with CMV peptide-loaded CBH 5477 cells in the continuous presence or absence of asunaprevir. Error bars represent standard deviation (n=3). Data are representative of two independent experiments.

[0028] [Figure 7-2]Figure 7 shows the adjustment of the repressive switch by N-terminal modification. Figures 7A-B are graphs of primary human T cells modified with HLA class I restriction CDK4 TCR and Zap70-PD1-SMASh, N-terminal alanine-added (modified) tuZap70-PD1-SMASh, or vector control, pretreated with 10 μM asunaprevir or DMSO control. The data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ EGFP high CD8+ (Figure 7A)+ T cells and CD4+ (Figure 7B)+ T cells in co-culture with NKIRTIL006 tumor cells in the continuous presence or absence of asunaprevir. Notably, the tuZap70-PD1-SMASh switch results in nearly equivalent inhibition of CD4+ T cell function in the absence of asunaprevir, while significantly increasing the recovery of T cell function in the presence of asunaprevir. Figures 7C–D are graphs of primary human T cells modified with either HLA class II restriction CMV TCR and tuZap70-PD1-SMASh (Figure 7C) or a vector control (Figure 7D), pretreated with 10 μM asunaprevir or DMSO control. The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CD4+ CMV TCR+ EGFP hyper-T cells co-cultured with CMV peptide-loaded CBH 5477 cells in the continuous presence or absence of asunaprevir. Error bars represent standard deviation (n=3). Data are representative of two independent experiments.

[0029] [Figure 8]Figure 8 shows different ITIM / ITSMs that include inhibitory tails that can control the activity of TCR T cells used in the CRASH-IT platform. Primary human T cells modified in a CRASH-IT embodiment containing HLA class I restriction CDK4 TCR and Zap70 (2xSH2)-X-SMASh, where X may be without an inhibitory tail, or the inhibitory tail (or cytoplasmic domain / intracellular domain / terminal domain) of PD1, BTLA, SIRPA, sialic acid-binding immunoglobulin-like lectin (Sigrec) 5, Sigrec 9, Sigrec 11, PECAM1, or LY9, or the vector control may be pre-treated with 10 μM asunaprevir or DMSO control. Figure 8A shows the location of the EGFP intermediate and EGFP high-gating in T cells. The data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ EGFP intermediate (Figure 8B-C) and high EGFP (Figure 8D-E), CD8+ (Figure 8B, Figure 8D), and CD4+ (Figure 8C, Figure 8E)+ T cells co-cultured with NKIRTIL006 tumor cells in or without asunaprevir. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0030] [Figure 9]Figure 9 shows that different ITIM / ITSMs, including inhibitory tails, can be used in the CRASH-IT platform to control CAR T cell activity. Primary human T cells modified with a CRASH-IT embodiment containing a second-generation anti-CD19-CD28-CD3ζ CAR and Zap70 (2xSH2)-X-SMASh, where X may be without an inhibitory tail, or the inhibitory tail (or cytoplasmic / intracellular / terminal domain) of PD1, BTLA, SIRPA, Siglec 5, Siglec 9, Siglec 11, PECAM1, or LY9, or the vector control may be pre-treated with 10 μM asunaprevir or DMSO control. Figure 9A shows the location of the EGFP intermediate and EGFP high-gating in T cells. The data depict intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+ EGFP intermediate (Figure 9B-C) and EGFP high (Figure 9D-E), CD8+ (Figure 9B, Figure 9D)+ T cells and CD4+ (Figure 9C, Figure 9E)+ T cells co-cultured with Daudi tumor cells in or without asunaprevir. Error bars represent the standard deviation (n=3). The data are representative of two independent experiments.

[0031] [Figure 10] Figure 10 shows that another SH2-containing docking domain can be used in the CRASH-IT platform. Primary human T cells modified with HLA class I restriction CDK4 TCR and Zap70 (2xSH2)-PD1 tail-SMASh, Syk (2xSH2)-PD1 tail-SMASh, Lck (SH4-Unique-SH3-SH2)-PD1 tail-SMASh, or vector controls were pretreated with 10 μM asunaprevir or DMSO control. Data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CD8+ CDK4 TCR+ EGFP hyper-T cells co-cultured with NKIRTIL006 tumor cells in or without asunaprevir. Error bars represent standard deviation (n=3). Data are representative of two independent experiments.

[0032] [Figure 11] Figure 11 shows a comparison of the immunoreceptor tyrosine-based switch motif (ITSM, bold) and immunoreceptor tyrosine-based inhibitory motif (ITIM, underlined) sequences in the cytoplasmic domains of PD1, BTLA, SIRPA, Siglec 5, Siglec 9, Siglec 11, PECAM1, and LY9. The common sequence for ITSM is TxYxxV / I, while the common sequence for ITIM is S / I / V / LxYxxI / V / L.

[0033] [Figure 12] Figure 12 shows a comparison of the activation motif (ITAM, bold and underlined) sequences based on immunoreceptor tyrosine in the CD3 ζ chain, CD3 ε chain, CD3 δ chain, CD3 γ chain, the γ (gamma) chain of the immunoglobulin receptor FcεRI, and DAP12. The common ITAM sequence is YxxI / Lx(6-8)YxxI / L.

[0034] [Figure 13] Figure 13 shows the regulation of NK cell function by CRASH-IT. Human NK cell line KHYG-1 modified with the Zap70-PD1-FKBP12F36V switch or vector control was pretreated with 0.5 μM dTAG-13 PROTAC or DMSO control. Figure 13A shows the location of EGFP intermediate gating in NK cells. Data depict intracellular IFNγ, IL2, and TNFα expression of EGFP intermediate NK cells in co-culture with K562 tumor cells (Figure 13B) or in the absence of K562 cells (Figure 13C), and in the continuous presence or absence of dTAG-13. Error bars represent the standard deviation (n=3). Data are representative of two independent experiments.

[0035] [Figure 14-1]Figure 14 shows that the CRASH-IT switch can be combined with various ITAM-containing CARs. (Figure 14A) Human primary T cells modified with Zap70-PD1-FKBP12F36V or (Figure 14B) a vector control and CD19 ScFv-CD28 (Hinge+TM)-CD3 ζ chain, CD19 ScFv-CD28 (Hinge+TM)-FCER1G, CD19 ScFv-CD3 ε chain (full length), CD19 ScFv-CD28 (Hinge+TM)-CD3 ε chain, or CD19 ScFv-CD28 (Hinge+TM)-DAP12 CARs were pretreated with 0.5 μM dTAG-13 or DMSO control. The data in Figures 14A-B show the intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CAR+, EGFP-heavy CD8+ T cells co-cultured with Daudi tumor cells in the continuous presence or absence of dTAG-13. The error bars represent the standard deviation (n=3).

[0036] [Figure 14-2] Figure 14 shows that the CRASH-IT switch can be combined with various ITAM-containing CARs. (Figure 14A) Human primary T cells modified with Zap70-PD1-FKBP12F36V or (Figure 14B) a vector control and CD19 ScFv-CD28 (Hinge+TM)-CD3 ζ chain, CD19 ScFv-CD28 (Hinge+TM)-FCER1G, CD19 ScFv-CD3 ε chain (full length), CD19 ScFv-CD28 (Hinge+TM)-CD3 ε chain, or CD19 ScFv-CD28 (Hinge+TM)-DAP12 CARs were pretreated with 0.5 μM dTAG-13 or DMSO control. The data in Figures 14A-B show the intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CAR+, EGFP-heavy CD8+ T cells co-cultured with Daudi tumor cells in the continuous presence or absence of dTAG-13. The error bars represent the standard deviation (n=3).

[0037] [Figure 15]Figure 15 is a schematic diagram of the Zap70-PD1-zinc finger degron switch (an example of an embodiment of the present invention that employs a CRBN polypeptide substrate domain capable of binding to a CRBN protein in response to a drug, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide) in the absence of IMiD (Figure 15A) or in the presence of IMiD (Figure 15B). Figure 15A shows that in the absence of IMiD such as thalidomide, lenalidomide, and pomalidomide, the Zap70-PD1-zinc finger fusion protein is stable and inhibits T cell function. Figure 15B shows that in the presence of IMiD, the fusion protein is degraded by the recruitment of CRBN E3 ligase, resulting in the restoration of T cell activity.

[0038] [Figure 16-1] Figure 16 shows that IMiD volume settings for CRASH-IT switch-expressing CD8 cells based on zinc finger degrons revealed designs with different drug sensitivities. Figure 16A shows the amino acid sequences of the zinc finger degrons used. The top zinc finger degron is derived from the IKZF1 ZF2-3 sequence. By replacing the β-turn sequence of IKZF1 ZF2 (within the rectangle) with the β-turn sequence of ZNF653 ZF4, ZFP91 ZF4, ZNF276 ZF4, or ZNF827 ZF1, we were able to create composite zinc finger sequences with improved IMiD sensitivity, as shown below. Individual C2H2 zinc finger sequences are underlined. Human primary T cells modified with CDK4 TCR and Zap70-PD1-zinc finger degron were pretreated with specified concentrations of lenalidomide (Figure 16B), pomalidomide (Figure 16C), and thalidomide (Figure 16D). The data show the intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP-highly expressing CD8+ T cells in co-culture with NKIRTIL006 tumor cells in the continuous presence of specified concentrations of lenalidomide, pomalidomide, or thalidomide. Error bars represent the standard deviation (n=2).

[0039] [Figure 16-2]Figure 16 shows that IMiD volume settings for CRASH-IT switch-expressing CD8 cells based on zinc finger degrons revealed designs with different drug sensitivities. Figure 16A shows the amino acid sequences of the zinc finger degrons used. The top zinc finger degron is derived from the IKZF1 ZF2-3 sequence. By replacing the β-turn sequence of IKZF1 ZF2 (within the rectangle) with the β-turn sequence of ZNF653 ZF4, ZFP91 ZF4, ZNF276 ZF4, or ZNF827 ZF1, we were able to create composite zinc finger sequences with improved IMiD sensitivity, as shown below. Individual C2H2 zinc finger sequences are underlined. Human primary T cells modified with CDK4 TCR and Zap70-PD1-zinc finger degron were pretreated with specified concentrations of lenalidomide (Figure 16B), pomalidomide (Figure 16C), and thalidomide (Figure 16D). The data show the intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression of CDK4 TCR+, EGFP-highly expressing CD8+ T cells in co-culture with NKIRTIL006 tumor cells in the continuous presence of specified concentrations of lenalidomide, pomalidomide, or thalidomide. Error bars represent the standard deviation (n=2).

[0040] [Figure 17-1]Figure 17 shows that the CRASH-IT switch based on the combined ZFP91 / IKZF1 (double ZF) is highly effective in restoring T cell function in the presence of IMiDs. Figure 17A shows the amino acid sequences of the zinc finger degrons used in the experiment. Wild-type zinc finger degrons derived from IKZF1, IKZF3, ZFP91, ZNF276, ZNF653, ZNF692 (all double ZF), or combined ZFP91 / IKZF1 zinc finger degrons with IKZF1 ZF3 (double ZF and single ZF, respectively) were used in the experiment. Individual C2H2 zinc finger sequences are underlined. Primary human T cells modified with Zap70-PD1-zinc finger degron, labeled as CDK4 TCR, were pretreated with 0.5 μM pomalidomide, 0.5 μM thalidomide, or DMSO control. The data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+, EGFP-heavy CD8+ T cells co-cultured with NKIRTIL006 tumor cells in the presence or absence of pomalidomide or thalidomide. Error bars represent the standard deviation (n=2).

[0041] [Figure 17-2]Figure 17 shows that the CRASH-IT switch based on the combined ZFP91 / IKZF1 (double ZF) is highly effective in restoring T cell function in the presence of IMiDs. Figure 17A shows the amino acid sequences of the zinc finger degrons used in the experiment. Wild-type zinc finger degrons derived from IKZF1, IKZF3, ZFP91, ZNF276, ZNF653, ZNF692 (all double ZF), or combined ZFP91 / IKZF1 zinc finger degrons with IKZF1 ZF3 (double ZF and single ZF, respectively) were used in the experiment. Individual C2H2 zinc finger sequences are underlined. Primary human T cells modified with Zap70-PD1-zinc finger degron, labeled as CDK4 TCR, were pretreated with 0.5 μM pomalidomide, 0.5 μM thalidomide, or DMSO control. The data show intracellular IFNγ, IL2, TNFα, and cell surface LAMP1 expression in CDK4 TCR+, EGFP-heavy CD8+ T cells co-cultured with NKIRTIL006 tumor cells in the presence or absence of pomalidomide or thalidomide. Error bars represent the standard deviation (n=2). [Modes for carrying out the invention]

[0042] Reference to sequence list The sequence listings, which are part of this disclosure, include text files containing nucleotide and / or amino acid sequences of the present invention. The subject matter of the sequence listings is incorporated herein in its entirety. Information recorded in computer-readable format is identical to the written sequence listings.

[0043] definition The headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described.

[0044] This disclosure contains copyrighted material (including figures, photographs of apparatus, or other aspects of this submission that are or may be copyrighted in any jurisdiction). The copyright holder has no objection to any facsimile copies of the patent documents or patent disclosures contained in the patent files or records of the Japan Patent Office, but reserves all other copyrights.

[0045] Various terms relating to the methods, compositions, uses, and other embodiments of the present invention are used throughout this specification and the claims. Unless otherwise specified, such terms should be given the common meaning in the art to which the invention pertains. Other specifically defined terms shall be construed in a manner consistent with the definitions provided herein. Any methods and materials similar or equivalent to those described herein may be used in carrying out the tests of the present invention, but preferred materials and methods are described herein. For the purposes of this invention, the following terms are defined.

[0046] As used herein, the singular terms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. For example, "a cell" includes a combination of two or more cells.

[0047] In this specification, the term "and / or" means a situation in which one or more of the described cases may occur individually or in combination with at least one of the described cases, up to a maximum of all of the described cases.

[0048] As used herein, the term “at least” means a specific value greater than or equal to that specific value. For example, “at least 2” is interpreted as “2 or greater,” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term “at most” means a specific value less than or equal to that specific value. For example, “at most 5” is interpreted as “5 or less,” i.e., 5, 4, 3, ... -10, -11, etc.

[0049] As used herein, the term “contains” is to be construed as inclusive, unrestricted, and non-exclusive. Specifically, this term and its variations mean that the specified structure, process, or component is included. These terms are not to be construed as excluding the existence of other structures, processes, or components. They also encompass the more restrictive term “consisting of.”

[0050] As used herein, “prior art” or “methods known to those skilled in the art” refers to situations in which the methods of the prior art used in the methods of the present invention are obvious to those skilled in the art. Practices of the prior art in molecular biology, biochemistry, cell culture, genomics, sequencing, medicine, pharmacology, immunology and related fields are well known to those skilled in the art and have been discussed in various handbooks and literature references.

[0051] In this specification, the term “exemplary” means “serving as an example, illustration, or illustration,” and should not be construed as excluding other configurations disclosed herein.

[0052] In this specification, the term “cancer” means a physiological condition in mammals typically characterized by uncontrolled cell proliferation. The terms “cancer,” “neoplasm,” and “tumor” are often used interchangeably to refer to cells that have undergone malignant transformation, becoming pathological to the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to those skilled in the art. As used herein, cancer cells include not only primary cancer cells but also cancer cells derived from such primary cancer cells, such as metastatic cancer cells, and cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors or hematological malignancies. Examples of cancer include, but are not limited to, leukemia, lymphoma, sarcoma, and carcinoma (e.g., colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, melanoma, lymphoma, non-Hodgkin lymphoma, colon cancer, (malignant) melanoma, thyroid cancer, papillary thyroid cancer, lung cancer, non-small cell lung cancer, and lung adenocarcinoma). As is well known, tumors can metastasize from their original site to one or more other body tissues or sites. When discussing treatment for a “neoplasm,” “tumor,” or “cancer” in a patient, it should include treatment for the primary cancer and, where appropriate, treatment for metastases.

[0053] As used herein, the terms “chimeric gene” or “chimeric nucleic acid” refer to any gene or nucleic acid not typically found in nature within the same species, in particular a gene or nucleic acid in which one or more parts of the nucleotide sequence are not related to each other in nature. For example, a promoter may not be related in nature to some or all of a transcription region or to another regulatory region, or different parts of a transcription region may not be related in nature. The term “chimeric gene” shall include an expression construct in which a promoter or transcription regulatory sequence is operably ligated to one or more coding sequences. Chimeric genes of chimeric nucleic acids are used in some embodiments to create chimeric proteins.

[0054] In this specification, the terms “protein” and “polypeptide” refer to molecules consisting of chains of amino acids, regardless of their specific mechanism of action, size, three-dimensional structure, or origin. Therefore, “fragments,” “parts,” or “segments” of polypeptides may still be referred to as “polypeptides.” “Isolated protein” or “isolated polypeptide” is used to refer to a protein or polypeptide that is no longer present in its natural environment, for example, in laboratory equipment or recombinant host cells.

[0055] As used herein, the terms “chimeric polypeptide,” “chimeric protein,” or “fusion protein” refer to any polypeptide of the same species that does not normally occur in nature, and in particular, a polypeptide in which one or more parts of the amino acid sequence are not related to each other in nature. For example, a chimeric polypeptide may include an N-terminal portion consisting of a first sequence of amino acids that are not related to each other in nature, and a C-terminal portion consisting of a second sequence of amino acids that are not related to each other in this order in nature. Chimeric polypeptides can be obtained, for example, from the transcription and translation of chimeric genes of nucleic acids.

[0056] As used herein, the terms “nucleic acid” or “polynucleotide” refer to any polymer or oligomer of (sequential) nucleotides. Nucleic acids may be DNA or RNA, or mixtures thereof, and may exist permanently or transiently in single-stranded or double-stranded forms, including homo-double-stranded, hetero-double-stranded, and composite states. The present invention intends any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of their bases. Polymers or oligomers may be heterogeneous or homogeneous in composition, may be isolated from naturally occurring sources, or may be manufactured artificially or synthetically. Accordingly, the term “isolated” means isolated from naturally occurring sources or manufactured artificially or synthetically.

[0057] As used herein, the term “pharmaceutical composition” means a pharmaceutically acceptable composition comprising a therapeutically effective amount of material together with one or more pharmaceutically acceptable carriers (additives) and / or diluents.

[0058] In this specification, the term “treatment” or “procedure” means the treatment of a tumor with therapeutic agents (including biological materials and cells), i.e., therapeutic drugs. Treatment may involve the administration of multiple drugs. Drugs may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition being treated. For example, treatment may be a combination therapy involving the administration of two drugs / medicines, one or more of which may be intended to treat the tumor. A treatment plan may be a predetermined schedule, plan, method or schedule of therapeutic administration, prepared by a physician or practitioner and adapted to the patient requiring treatment. A treatment plan may indicate one or more of the following: the type of treatment to be administered to the patient, the dosage of each drug, the time interval between administrations, the length of each treatment, and, if there are drug-free periods, the number and nature of those periods. In the case of combination therapy, a single treatment plan may be provided that indicates the method of administration of each drug.

[0059] As used herein, the terms “patient,” “individual,” or “subject” refer to mammals. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the patient, individual, or subject is a human. In some embodiments, the patient may be a “cancer patient,” that is, a person who has or is at risk of having one or more cancers.

[0060] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which they are introduced. Certain vectors can induce the expression of the nucleic acid to which they are operatively ligated. In this specification, such vectors are referred to as "expression vectors."

[0061] Detailed description of the invention Any method, use, or composition described herein is intended to be applicable in relation to any other method, use, or composition described herein. Embodiments described in the context of the methods, uses, and / or compositions of the present invention can be adopted in relation to any other method, use, or composition described herein. Thus, embodiments relating to a certain method, use, or composition can also be applied to other methods, uses, and compositions of the present invention.

[0062] As embodied and broadly described herein, the present invention is directed toward the remarkable discovery that it is now possible to control T cell activity, particularly the cytotoxic activity of T cells, and / or cytokine secretion by such T cells, e.g., CD4 or CD8-positive T cells, and natural killer cell activity, particularly the cytotoxic activity of NK cells, and / or cytokine secretion by such NK cells, using chimeric polypeptides as disclosed herein.

[0063] More specifically, the inventors have developed an innovative system for modulating and / or manipulating signaling pathways in T cells and NK cells. This system can modulate T cell activity in a time- and / or dose-dependent manner as a result of signaling mediated by T cell receptors (TCRs) and / or chimeric antigen receptors (CARs), and can modulate NK cell activity in a time- and / or dose-dependent manner as a result of signaling mediated by NK cell receptors (NKRs) and / or chimeric antigen receptors (CARs). The system can be suitably used with any cells expressing TCRs and / or CARs, including native T cells or T cells engineered to express specific (modified) TCRs and / or CARs, as well as any cells expressing NKRs and / or CARs, including native or engineered NK cells. Accordingly, according to embodiments of the present invention, the cells according to the present invention are lymphocytes, in particular T cells or NK cells. Such T cells and / or NK cells include any "modified" T cells or NK cells, such as CAR T cells, CAR NK cells, multiple CAR T cells, multiple CAR NK cells, tandem CAR T cells, tandem CAR NK cells, recombinant TCR T cells, and recombinant TCR NK cells.

[0064] This disclosure provides precise regulation of T cell and NK cell activity (e.g., cytotoxic activity and / or cytokine secretion) by the chimeric polypeptide according to the present invention through the presence of small molecule regulatory protein stability domains that are used to regulate (e.g., decrease or increase) the expression of the chimeric polypeptide according to the present invention in a time and / or dose-dependent manner.

[0065] This chimeric polypeptide is designed to interact with the activation motif (ITAM) based on phosphorylated immunotyrosine in the TCR / CD3 complex, including signaling molecules with ITAMs such as DAP12, the γ (gamma) chain of the immunoglobulin receptor FcεRI, or the CD3 ζ chain, as well as / or in the CAR and / or NK cell receptor (NKR) complex (Lanier et al, Nat Immunol. 2008 May; 9(5): 495-502).

[0066] The tyrosine residues within these ITAM motifs are phosphorylated after interaction with the receptor molecule and its ligand, forming docking sites for other proteins involved in cellular signaling pathways. Interaction between the chimeric polypeptide according to the present invention and TCR and / or CAR inhibits T cell activation (and subsequent cytotoxic effects and / or cytokine secretion). Similarly, interaction between the chimeric polypeptide according to the present invention and NKR and / or CAR in NK cells inhibits NK cell activation (and subsequent cytotoxic effects and / or cytokine secretion).

[0067] In a preferred embodiment, the chimeric polypeptide includes, following the small molecule regulatory protein stability domain and the domain that interacts with the phosphorylated ITAM motif, an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based suppressor motif (ITIM). Such motifs are, for example, located in the inhibitory tail of PD1 and have been suggested to be involved in the immunosuppressive effect of PD1 (Boussiotis et al, Cancer J. 2014 Jul-Aug; 20(4): 265-271). It was unexpectedly discovered that the presence of such an ITSM, preferably such an ITSM and ITIM, in the chimeric polypeptide allows the chimeric polypeptide to effectively inhibit signal transduction by, for example, TCR, NKR, or CAR (when a ligand binds to its receptor). Here, we demonstrate that these ITSMs, preferably ITSMs and ITIMs present in the inhibitory tail of inhibitory immune receptor proteins such as PD1, can inhibit TCR and / or CAR signaling in T cells and NKR signaling in NK cells without requiring the presence of the extracellular domain of the inhibitory protein or interaction with its ligand (e.g., PD-L1 for PD1). Combined with small molecule regulatory protein stability domains that enable dose-dependent expression of chimeric polypeptides in cells, e.g., T cells, this system provides an efficient and reliable method for precisely controlling T cell function, such as T cell activation, T cell cytotoxicity, and / or T cell cytokine secretion. In this way, T cell function can be prevented / suppressed or activated, and T cell functionality can be maintained, restored, safe, and controllable both in vitro and in vivo (i.e., in the treatment of cancer or other conditions such as autoimmune diseases that rely on the use of T cells, including T cells expressing (modified) TCRs and / or CARs).

[0068] Similarly, NK cell function can be prevented / suppressed or activated, and its functionality can be maintained, restored, and safely controlled both in vitro and in vivo (i.e., in the treatment of cancer or other conditions such as autoimmune diseases that rely on the use of NK cells, including NK cells expressing (modified) NKR and / or CARs).

[0069] Therefore, according to a first aspect of the present invention, a cell is provided comprising a chimeric polypeptide, or a nucleic acid comprising a polynucleotide encoding the chimeric polypeptide. The chimeric polypeptide is a) A first portion comprising the SH2 domain derived from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), and b) Includes a second portion containing a small molecule regulatory protein stability domain.

[0070] The cells according to the present invention may be any cells that can suitably contain a chimeric polypeptide as disclosed herein or a nucleic acid encoding such a chimeric polypeptide. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. Preferably, the cells are eukaryotic cells, and more preferably, mammalian cells such as human cells. The cells may be specialized cells such as T cells or NK cells, or any other type of cell, including (undifferentiated) stem cells.

[0071] The polypeptide according to the present invention is a chimeric polypeptide comprising at least a first and a second portion. The order in which the first and second portions are present in the chimeric peptide is not important. The present invention is not limited by the position of the first, second, or, in some embodiments, a third portion in the chimeric polypeptide. For example, in some embodiments, the first or second portion may be fused (e.g., genetically linked) to the N-terminus or C-terminus of the chimeric polypeptide, or may exist within the chimeric polypeptide. In other words, in the chimeric polypeptide, the first and / or second portions may be located at the C-terminus, at the N-terminus, or sandwiched between additional portions at the C-terminus and / or N-terminus. The first portion may be more C-terminal than the second portion, or more N-terminal than the second portion.

[0072] The first and second parts may be directly adjacent to each other, or they may be separated from each other by an additional part, i.e., an additional (sequence) of amino acid residues.

[0073] The chimeric polypeptides disclosed herein are characterized by the presence of a first portion comprising a protein-derived SH2 domain that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM). As can be seen from the examples, the SH2 domain may be any protein-derived SH2 domain capable of binding to a phosphorylated ITAM. Those skilled in the art will be familiar with suitable SH2 domains used in chimeric polypeptides such as those disclosed herein and / or can readily identify such suitable SH2 domains or proteins containing such SH2 domains capable of binding to phosphorylated ITAMs. As those skilled in the art will know, suitable SH2 domains are selected, for example, with consideration to ITAMs contained in the TCR / CD3 complex and / or CAR and / or NK cell receptor (NKR) complexes on which the chimeric polypeptide according to the present invention is designed. In other words, the chimeric proteins according to the present invention comprise an SH2 domain from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), the ITAM comprising, for example, a TCR or CAR complex to be targeted in the context of the present invention.

[0074] In this specification, the term "SH2 domain" refers to the SRC homologous 2 domain. The SH2 domain is a structurally conserved protein domain found in Src oncoproteins and many other intracellular signal transduction proteins. Proteins possessing the SH2 domain can dock with phosphorylated tyrosine residues of other proteins. Thus, the SH2 domain is a modular protein domain that functions as an adapter, binding to the phosphorylated peptides of its respective protein binding partners and mediating protein-protein interactions.

[0075] SH2 domains typically bind to phosphorylated tyrosine residues within long peptide motifs in target proteins. While SH2 domains do not possess intrinsic catalytic activity, they play a role in localizing the bound functional domain within the polypeptide to the vicinity of the appropriate substrate, activator, or inhibitor (Ngoenkam et al, Immunology. 2018 Jan; 153(1): 42-50).

[0076] Some SH2 domains interact with proteins that have an activation motif (ITAM) based on phosphorylated immunoreceptor tyrosine, while others may interact with proteins that have an inhibitory motif (ITIM) based on phosphorylated immunoreceptor tyrosine. In this invention, SH2 domains derived from proteins that bind to the activation motif (ITAM) of phosphorylated immunoreceptor tyrosine are used. SH2 domains from proteins that bind to phosphorylated ITAM are important for the regulation of T cell activity or NK cell activity by chimeric peptides as disclosed herein, but it was surprisingly found that SH2 domains that interact with ITIM are not at all or very suitable for use in chimeric polypeptides according to the present invention.

[0077] In preferred embodiments, the SH2 domain is derived from a protein that binds phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the TCR complex, NKR complex, and / or CAR.

[0078] In some embodiments, the SH2 domain is the SH2 domain annotated as βA-αA-βB-βC-βD-βE-βF-αB-βG for 120 known human SH2 domains by Liu et al. (Mol Cell. 2006;22(6):851-868. doi:10.1016 / j.molcel.2006.06.001). (β refers to the β chain, and α refers to the α helix) (Eck et al, Nature. 1993;362(6415):87-91. doi:10.1038 / 362087a0). The SH2 domain can be found in various databases well known to those skilled in the art (see, for example, smart.embl.de / smart / do_annotation.pl?DOMAIN=SM00252 or www.ebi.ac.uk / interpro / entry / InterPro / IPR000980 / ).

[0079] The term "immune receptor tyrosine activation motif (ITAM)" used here refers to a conserved sequence of four amino acids that is repeated twice and found in the cytoplasmic tail (i.e., terminal domain) of specific cell surface proteins of the immune system. A semi-ITAM is one in which a tyrosine residue (Y) is separated from a leucine residue (L) or isoleucine residue (I) by any two amino acids. The common sequence of semi-ITAMs is YxxL / l. Two semi-ITAMs are usually separated by 6-8 amino acids to form a complete ITAM. The common sequence of an ITAM is YxxL / lx(6-8)YxxL / l. ITAMs play a crucial role in immune cell signaling and are found in the cytoplasmic tail of T cell receptor complexes (CD3 ε chain, CD3 δ chain, CD3 γ chain, CD3 ζ chain). In NK cells, ITAM is present in the NK cell receptor complex, which includes the CD3 ζ chain, the γ (gamma) chain of the immunoglobulin receptor FcεRI, and DAP12 (Lanier et al, Nat Immunol. 2008 May; 9(5): 495-502). ITAM is also present in chimeric antigen receptor (CAR) complexes, including the CD3 ζ chain (Abate-Daga et al, Mol Ther Oncolytics. 2016; 3: 16014), the CD3 ε chain (Nolan et al, Clin Cancer Res. 1999 Dec;5(12):3928-41), the γ (gamma) chain of the immunoglobulin receptor FcεRI (Ren-Heidenreich et al, Cancer Immunol Immunother. 2002 Oct;51(8):417-23), and DAP12 (Topfer et al, J Immunol. 2015 Apr 1;194(7):3201-12).

[0080] The tyrosine residues of the ITAM motif are phosphorylated after the receptor molecule interacts with its ligand, forming a docking site with other proteins involved in cellular signaling pathways. Once phosphorylated, proteins containing an SH2 domain that binds to such phosphorylated ITAM can interact with the phosphorylated ITAM present, for example, on the CD3 ζ chain. Several proteins are known to contain terminal domains with one or more ITAM motifs. Examples of such proteins include the CD3 γ chain, CD3 δ chain and CD3 ε chain, CD3 ζ chain, the γ (gamma) chain of the immunoglobulin receptor FcεRI, and DAP12.

[0081] The chimeric polypeptides disclosed herein are further characterized by the presence of a second portion constituting a small molecule regulatory protein stability domain. This small molecule regulatory protein stability domain is sometimes referred to as a controllable destabilization domain. This small molecule regulatory protein stability domain or controllable destabilization domain in the chimeric polypeptide of the present invention is used to regulate (e.g., decrease or increase) the expression of the chimeric polypeptide of the present invention in a time and / or dose-dependent manner.

[0082] More specifically, the small molecular weight regulatory protein stabilization domain or controllable destabilization domain in the chimeric polypeptide of the present invention is a domain that is controlled by supplying a compound (e.g., a small molecule) to the cells constituting the chimeric polypeptide. Upon addition of the compound, the small molecular weight regulatory protein stabilization domain or controllable destabilization domain in the chimeric polypeptide is modified to induce degradation of the chimeric polypeptide.

[0083] In other words, in response to such compounds, the chimeric polypeptide according to the present invention is degraded through interaction between the compound and the small molecule regulatory protein stabilization domain or controllable destabilization domain in the chimeric polypeptide, thereby reducing the level, i.e., concentration, of the chimeric polypeptide in the cell. Consequently, the reduction in the level, i.e., concentration, of the chimeric polypeptide in the cell reverses the inhibition of T cell function, e.g., T cell activation, T cell toxicity, and / or T cell cytokine secretion, as a result of signaling via TCR and / or CAR.

[0084] Similarly, in NK cells, a decrease in the intracellular level, or concentration, of the chimeric polypeptide reverses NK cell function, such as NK cell activation, NK cell toxicity, and / or suppression of cytokine secretion by NK cells, as a result of NKR and / or CAR-mediated signaling. In this way, T cell function and / or NK cell function are regulated by the chimeric polypeptide of the present invention. In this way, by supplying cells with compounds that interact with the small molecule regulatory protein stabilization domain or controllable destabilization domain in the chimeric polypeptide, T cell function and / or NK cell function can be regulated by causing degradation of the chimeric polypeptide of the present invention.

[0085] Unexpectedly, the chimeric polypeptide of the present invention was found to reversibly and / or dose-dependently regulate T cell function or NK cell function via the small molecular regulatory protein stabilization domain or controllable destabilization domain within the chimeric polypeptide (see Examples). In the absence of compounds that interact with the small molecular regulatory protein stabilization domain or controllable destabilization domain and thereby cause disruption of the chimeric polypeptide, the chimeric polypeptide of the present invention suppresses T cell function by inhibiting signal transduction via the TCR and / or CAR as a result of the interaction between the chimeric polypeptide and the TCR / CD3 complex and / or CAR.

[0086] Similarly, in the absence of compounds that interact with small molecule regulatory protein stability domains or controllable destabilization domains and cause degradation of the chimeric polypeptide, the chimeric polypeptide of the present invention interacts with the NKR complex, which includes the CD3 ζ chain, the γ (gamma) chain of the immunoglobulin receptor FcεRI, and signaling molecules with ITAM such as DAP12 and / or CAR, thereby inhibiting NK cell function by inhibiting signaling via NKR and / or CAR. In the presence of such compounds, the chimeric polypeptide is induced into the intracellular proteolytic system, thereby resolving the inhibition of T cell function and / or NK cell function. This resolving of the inhibition of T cell function and / or NK cell function is dose-dependent, as demonstrated by the examples.

[0087] Alternatively, a small molecule regulatory protein stabilization domain or a controllable destabilization domain may be used to induce the degradation of the chimeric protein of the present invention in the absence of the small molecule. In such embodiments, the presence of the small molecule stabilizes the chimeric protein, thereby impairing the function of T cells or NK cells, and the removal of the small molecule compound restores the activity of T cells or NK cells.

[0088] The present invention is not limited to any small molecule controlled protein stabilization domain or controllable destabilization domain. In fact, any small molecule controlled protein stabilization domain or controllable destabilization domain that confers stability to a chimeric polypeptide may be used in the present invention (Roth et al, Cell Mol Life Sci. 2019 Jul;76(14):2761-2777). Here, chimeric polypeptide degradation occurs when the small molecule controlled protein stabilization domain or controllable destabilization domain in the chimeric polypeptide is modified to direct the chimeric polypeptide toward degradation by the presence of its homologous small molecule. Those skilled in the art are well aware of such suitable small molecule controlled protein stabilization domains or controllable destabilization domains. A non-limiting example of a small molecule controlled protein stabilization domain or controllable destabilization domain is a self-cleaving degron (SED), which comprises an inhibitory protease, a homologous cleavage site, and a degron sequence (Chung et al, Nat Chem Biol. 2015 Sep;11(9):713-20) or proteolytic target chimeric (Protac) binding domain. The Protac that can sequentially bind to the Protac-binding domain in the chimeric polypeptide according to the present invention includes an E3 ubiquitin ligase binding group (E3LB), a linker, and a protein binding group that binds to the Protac-binding domain in the chimeric polypeptide (Nabet et al, Nat Chem Biol. 2018 May;14(5):431-441, An et al, EBioMedicine. 2018 Oct; 36: 553-562). A preferred example of the small molecular weight regulatory protein stability domain or controllable destabilization domain used in the present invention is the CRBN polypeptide substrate domain, which can bind to the CRBN protein in response to a drug and thereby promote ubiquitin pathway-mediated degradation of the chimeric polypeptide. A typical example of such a small molecular weight regulatory protein stability domain is the so-called zinc finger degron.

[0089] Small molecule IMiDs such as thalidomide, lenalidomide, and pomalidomide recruit these Cys2-His2 (C2H2) zinc finger domain-containing proteins to the CRL4CRBN E3 ubiquitin ligase substrate receptor cereblon (CRBN), in vivo, inducing ubiquitination and proteasomal degradation of transcription factors such as IKZF1 and ioloz (IKZF3). When IMiDs are provided to cells expressing heterologous proteins containing such zinc finger domains, these zinc finger domains can be used both in vitro and in vivo to target heterologous proteins for degradation in a time- and dose-dependent manner (see Koduri et al PNAS (2019) 116 (7) 2539-2544; doi.org / 10.1073 / pnas.1818109116).

[0090] In fact, numerous possible zinc finger polypeptides and zinc finger domains have been incorporated into the degradation of target proteins via small molecules, for example, IMiDs (thalidomide, lenalidomide, pomalidomide, etc.) (see, e.g., Sievers et al. Science. 2018 Nov 2; 362(6414): eaat0572.doi:10.1126 / science.aat0572). With this in mind, those skilled in the art are well aware that the use and design of CRBN polypeptide substrate domains capable of binding to CRBN proteins in response to drugs facilitates ubiquitin-mediated degradation of chimeric polypeptides. In particular, as detailed herein, CRBN polypeptide substrate domains are C2H2 zinc finger proteins or fragments thereof capable of drug-inducible binding to CRBN polypeptides.

[0091] According to a preferred embodiment, the present invention provides cells in which the chimeric polypeptide comprises a third portion comprising an immune receptor tyrosine-based switch motif (ITSM), an immune receptor tyrosine-based inhibitory motif (ITIM), or preferably an ITSM and an immune receptor tyrosine-based inhibitory motif (ITIM). According to another embodiment, the third portion comprises an immune receptor tyrosine-based switch motif (ITSM) and / or an immune receptor tyrosine-based inhibitory motif (ITIM), preferably an ITSM and an immune receptor tyrosine-based inhibitory motif (ITIM).

[0092] Preferably, the third portion includes an immune receptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immune receptor tyrosine-based inhibitory motif (ITIM).

[0093] The order in which the first, second, and third parts are located in the chimeric peptide is not important. The present invention is not generally limited by the position of the first, second, or third part in the chimeric polypeptide. For example, depending on the embodiment, the first, second, or third part may be fused (e.g., genetically linked) to the N-terminus or C-terminus of the chimeric polypeptide, or it may be located within the chimeric polypeptide. In other words, in the chimeric polypeptide, the first, second, or third part may be located at the C-terminus, at the N-terminus, or sandwiched between other parts at the C-terminus and / or N-terminus. Examples of preferred orders for the first part (P1), second part (P2), and third part (P3) are generally any of xP1xP2xP3x, xP1xP3xP2x, xP2xP1xP3x, xP2xP3xP1x, xP3xP1xP2x, or xP3xP2xP1x. Here, x at any position independently indicates the absence of additional amino acid residues, or the presence of one or more additional amino acid residues that do not form part of P1, P2, and / or P3. With respect to the order of the first and second parts, for example in embodiments where a third part is absent, and similar to the above example, examples of a suitable order of the first part (P1) and the second part (P2) may generally be xP1xP2x or xP2xP1x, where x at any position independently indicates the presence of additional amino acid residues, or the presence of one or more additional amino acid residues that do not form part of P1 and / or P2.

[0094] At the same time, those skilled in the art know that the first part may include, in addition to the SH2 domain from the protein that binds the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), an additional domain or amino acid, such as one or more amino acids that are normally adjacent to the SH2 domain (one or both sides) in the protein that binds the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM).

[0095] At the same time, those skilled in the art know that the second portion may contain additional domains or amino acids on one or both sides, in addition to the small molecule regulatory protein stability domain.

[0096] At the same time, those skilled in the art will know that the third part may include, in addition to an immune receptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immune receptor tyrosine-based inhibitory motif (ITIM), further domains or amino acids, such as one or more of the amino acids that typically flank these motifs (on one or both sides).

[0097] Those skilled in the art know that, as long as a small molecular weight regulatory protein stability domain or a controllable destabilization domain is positioned within the chimeric polypeptide when it comes into contact with a compound that interacts with the small molecular weight regulatory protein stability domain or the controllable destabilization domain, the first, second, and third portions of the chimeric polypeptide of the present invention are present in the chimeric polypeptide in any order, the chimeric polypeptide is degraded, and the inhibition of T cell function and NK cell function via interaction with TCRs and CARs is released.

[0098] The terms "ITIM" and "ITSM" are known to those skilled in the art (Liu et al, Mol Cell Proteomics. 2015 Jul; 14(7):1846-58).

[0099] As used herein, the term "immune receptor tyrosine system inhibitory motif (ITIM)" generally refers to a conserved amino acid sequence found in the cytoplasmic tail of many inhibitory receptors of the immune system. An ITIM motif comprises a serine residue (S), an isoleucine residue (I), a valine residue (V), or a leucine residue (L), separated from the tyrosine residue (Y) by any other amino acid residue (x), and then separated by any two other amino acids other than the isoleucine residue (I), valine residue (V), or leucine residue (L). The common symbol is S / l / V / LxYxxl / V / L. In vivo, inhibitory receptors containing ITIM interact with ligands, causing the ITIM motif to be phosphorylated by Src kinase enzymes. This recruits SH2-containing protein tyrosine phosphatases (PTPs) such as SHP-1 and SHP-2 (Coxon et al, Blood. 2017 Jun 29;129(26):3407-3418) and lipid phosphatases such as SHIP-1. PTPs counteract the positive regulatory effects of protein tyrosine kinases (PTKs) such as Lck and Zap70, thereby negatively regulating T cell signaling (Lorenz et al, Immunol Rev. 2009 Mar; 228(1): 342-359). By dephosphorylating ITAM on TCRs, CARs, and other immune receptors, PTPs can reverse the activating effect of ITAM phosphorylation. Lipid phosphatases regulate cell signaling by altering the concentrations of lipid phosphates and their dephosphorylated forms.

[0100] As used herein, the term “immune receptor tyrosine-based switch motif (ITSM)” refers to a sequence of conserved amino acids found in the cytoplasmic tail (or cytoplasmic domain, intracellular domain, or terminal domain) of many inhibitory receptors of the immune system, in other words, in the portion of a protein that is present in the cytoplasm of a cell (and not in the membrane and / or extracellular space). An ITSM motif contains a threonine residue (T) separated by a tyrosine residue (Y) separated by two other amino acids from a valine residue (V) or an isoleucine residue (I) and then by any other amino acid residue. The common symbol is TxYxxV / I. Similar to ITIM-containing inhibitory receptors, ITSM-containing inhibitory receptors interact with their ligands, and the ITIM motif becomes phosphorylated by the Src kinase enzyme, allowing for the recruitment of SH2-containing phosphorylated products such as SHP-1 and SHP-2 (Lorenz et al, Immunol Rev. 2009 Mar; 228(1): 342-359). Some studies have reported that both ITIM and ITSM motifs contribute to the inhibitory signaling of PD1 (Boussiotis et al, Cancer J. 2014 Jul-Aug; 20(4): 265-271, Peled et al, Proc Natl Acad Sci US A. 2018 Jan 16;115(3):E468-E477). On the other hand, other studies have shown that the ITSM motif is primarily involved in the inhibitory effect of PD1, while the ITIM motif has only a limited effect (Chemnitz et al, J Immunol. 2004 Jul 15;173(2):945-54, Yokosuka et al, J Exp Med. 2012 Jun 4;209(6):1201-17).

[0101] According to the present invention, ITSM and ITIM may originate from the same protein (e.g., PD1) or from two different proteins (e.g., ITIM derived from PD1 and ITSM derived from LY9). Preferably, ITSM and ITIM originate from or are obtained from the same protein. Preferably, ITSM and ITIM are separated from each other by 15 to 25 amino acids in the third portion of the chimeric polypeptide of the peptide of the present invention. In some embodiments, the amino acids separating ITSM and ITIM are the same amino acids in the protein from which ITSM and ITIM originate or are obtained.

[0102] Preferably, in the chimeric polypeptide according to the present invention, the first portion containing the SH2 domain from a protein that binds to an activation motif based on phosphorylated immunoreceptor tyrosine consists of at least 80 adjacent amino acids and / or at most 800 adjacent amino acids, preferably at least 100 adjacent amino acids and / or at most 400 amino acids, for example, 150 to 300 amino acids.

[0103] Preferably, in the chimeric polypeptide according to the present invention, the third portion comprising an immune receptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immune receptor tyrosine-based inhibitor motif (ITIM), consists of at least 30 adjacent amino acids and / or at most 600 adjacent amino acids, preferably at least 80 adjacent amino acids and / or at most 200 amino acids, for example, 85 to 190 amino acids.

[0104] Furthermore, cells according to the present invention are provided. The small molecule regulatory protein stability domain is selected from the group consisting of an inhibitory protease, a homogeneous cleavage site, and a self-cleaving degron (SED) containing a degron sequence, a proteolytic target chimeric (Protac) binding domain (Protac includes an E3 ubiquitin ligase binding group (E3LB), a linker, and a protein binding group that binds to the Protac binding domain in the chimeric polypeptide), and a CRBN polypeptide substrate domain that can bind to a CRBN protein in response to a drug, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide. For example, the protein binding group of Protac is the Protac binding domain in the chimeric polypeptide of the present invention (e.g., FKBP12 F36V AP1867, which binds to (SEQ ID NO: 35, Nabet et al. Nat Chem Biol. 2018 May;14(5):431-441), may also be used. For a recent review of PROTAC technology, see Zou et al. Current Protocols (2019) V37:1: pp 21 -30; doi.org / 10.1002 / cbf.3369.

[0105] As will be obvious to those skilled in the art, the small molecule regulatory protein stability domain (or controllable destabilization domain) may preferably be any domain that provides chimeric polypeptide stability such that degradation of the chimeric polypeptide occurs when the small molecule regulatory protein stability domain (or controllable destabilization domain) in the chimeric polypeptide is modified / targeted in such a way as to lead to degradation of the chimeric polypeptide by the presence of its homologous small molecule.

[0106] Preferably, the small molecule regulatory protein stability domain is a CRBN polypeptide substrate domain that can bind to the CRBN protein in response to a drug, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide, and preferably, the CRBN polypeptide substrate domain is a C2H2 zinc finger protein or a fragment thereof that can drug-inducibly bind to the CRBN polypeptide.

[0107] Preferably, the small molecule regulatory protein stability domain is a proteolytic target chimeric (Protac) binding domain. This domain can bind to a congener Protac.

[0108] Preferably, the small molecule regulatory protein stability domain is a self-cleaving degron (SED), which comprises an inhibitory protease, a homologous cleavage site, and a degron sequence. Such self-cleaving degrons are well known to those skilled in the art (Chung et al, Nat Chem Biol. 2015 Sep;11(9):713-20).

[0109] As used herein, the term “self-cleaving degron” (SED) means a polypeptide or protein complex comprising an inhibitory protease, a homogeneous cleavage site, and a degron sequence (or degradation sequence). A self-cleaving degron is part of the chimeric polynucleotide of the present invention, and the protease can cleave the chimeric polypeptide of the present invention to separate the degron sequence from the rest of the chimeric polypeptide.

[0110] In some embodiments, the cleavage of the chimeric polypeptide of the present invention by a protease separates at least a first portion of the chimeric polypeptide, including the SH2 domain from a protein that binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) (which is part of the second portion in the chimeric polypeptide) from the degron sequence.

[0111] In some embodiments, the cleavage of the chimeric polypeptide of the present invention by a protease separates at least a first portion of the chimeric polypeptide containing an SH2 domain from a protein that binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) from a degron sequence (which is part of the second portion in the chimeric polypeptide) from a third portion of the chimeric polypeptide of the present invention containing an immunoreceptor tyrosine-based switch motif (ITSM), preferably the ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM).

[0112] The protease itself may or may not be removed from the portion of the chimeric polypeptide that has been separated from the degron sequence.

[0113] As used herein, the term "degron" means a protein or a portion thereof that is important for regulating the rate of proteolysis. Various degrons known in the art, including but not limited to short amino acid sequences, structural motifs, and exposed amino acids, can be used in various embodiments of this disclosure. Degrons identified from various organisms can be used.

[0114] As used herein in the context of SED, the terms “degron sequence” or “degradation sequence” refer to sequences that promote the degradation of adherent proteins via either the proteasome pathway or the autophagocytosal-lysosome pathway. Many different degradation sequences / signals (e.g., those of the ubiquitin-proteasome system) are known in the art and any of them may be used as provided herein. For a discussion of degradation sequences and their functions in proteolysis, see, for example, Kanemaki et al, Pflugers Arch. 2013 Mar;465(3):419-25, and Erales et al, Biochim Biophys Acta. 2014 Jan;1843(1):216-21.

[0115] As used herein, the term “homogeneic cleavage site” means a specific sequence or sequence motif that is recognized and cleaved by the inhibitory proteases of SEDs. A protease cleavage site includes a specific amino acid sequence or motif that is recognized by the protease during proteolytic cleavage, and typically includes the vicinity of one to six amino acids on either side of a readily cleavable bond that is used to bind to the active site of the protease and be recognized as a substrate.

[0116] As used herein, the term “inhibitory protease” refers to a protease that is inactivated by the presence of a specific drug or compound, such as a small molecule compound (e.g., one that binds to a protease) (Leuw et al, GMS Infect Dis. 2017; 5: Doc08, Lv et al, HIV AIDS (Auckl). 2015; 7: 95-104). In some embodiments, the inhibitory protease is active (cleaves a homogeneous cleavage site) in the absence of a specific drug, but inactive (does not cleave a homogeneous cleavage site) in the presence of the specific drug. In some embodiments, the specific drug is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given inhibitory protease of this disclosure.

[0117] In embodiments, the SED is a small molecule-assisted blockade (SMASh) technology, i.e., a small molecule-assisted blockade tag (SMASh tag) (Chung et al, Nat Chem Biol. 2015 Sep;11(9):713-20). This includes a degradation signal (i.e., a degron sequence) and a protease cleavage site that cleaves the degron from the rest of the chimeric polypeptide of the present invention. However, in the presence of a protease inhibitor, this cleavage is blocked, and the degron can induce rapid degradation of the chimeric polypeptide. In some embodiments, the SMASh may include a hepatitis C virus-derived NS3 / 4A protease (inhibited by, for example, asunaprevir or grazoprevir) flanked by the degron domain that induces proteasomal degradation. HCV NS3 / 4A protease inhibitors include asunaprevir, grazoprevir, glecaprevir, boxilaprevir, paritaprevir, simeprevir, boceprevir, and telaprevir (Majumdar et al, Aliment Pharmacol Ther. 2016 Jun;43(12):1276-92, Ahmed et al, World J Hepatol. 2018 Oct 27; 10(10): 670-684).

[0118] In another embodiment, the small molecule controlled protein stability domain (or controllable destabilization domain) comprises a proteolytic target chimeric (Protac) binding domain, where Protac comprises an E3 ubiquitin ligase binding group (E3LB), a linker, and a protein binding group that binds to the Protac binding domain in the chimeric polypeptide.

[0119] One of the major degradation pathways in cells is the ubiquitin-proteasome system. In this system, when a protein is ubiquitinated, it is labeled for degradation by the proteasome. Protein ubiquitination is achieved by E3 ubiquitin ligases, which bind to proteins and attach ubiquitin molecules to them. E3 ubiquitin ligases are part of a pathway that includes E1 ubiquitin activators and E2 ubiquitin conjugates, which make ubiquitin available and allow E3 ubiquitin ligases to attach to proteins.

[0120] Protacs was developed to utilize this degradation pathway. Protacs binds the target protein to an E3 ubiquitin ligase. To promote proteasome-mediated protein degradation, Protac consists of a group that binds to the E3 ubiquitin ligase and a group that binds to the protein to be degraded (i.e., a Protac-binding domain in the polypeptide). These groups are usually linked by a linker. This molecular structure allows the E3 ubiquitin ligase and the target protein to be brought into close proximity, ubiquitinated, and labeled in a way that facilitates degradation.

[0121] As used herein, the term “Protac” generally refers to a proteolytic target chimeric molecule having three components: an E3 ubiquitin ligase binding group (E3LB), a linker, and a protein binding group. The Protacs and Protac-binding domains used in the chimeric polypeptide according to the present invention are well known to those skilled in the art (An et al, EBioMedicine. 2018 Oct; 36: 553-562).

[0122] As used herein, the term “linker” refers to a chemical moiety containing a chain of atoms that covalently bonds one component of a Protac to another component of that Protac. In various embodiments, linkers are typically 8 to 20 atoms long (see Cyrus et al, Mol Biosyst. 2011 Feb; 7(2): 10.1039 / c0mb00074d, Nabet et al, Nat Chem Biol. 2018 May; 14(5): 431-441). Commercially available linkers include, for example, those offered by Medchemexpress (www.medchemexpress.com).

[0123] A protein-binding group is a group that binds to the Protac-binding domain present in the target protein, in this case, a chimeric polypeptide. The protein-binding group may be any site that specifically binds to a protein (binds to the target protein), and includes, for example, many non-limiting examples of small target protein sites, namely Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting aryl hydrocarbon receptors (AHRs) (see US2014 / 0356322 and US2016 / 0045607).

[0124] In some embodiments, the protein-binding group in Protac is AP1867 (www.medchemexpress.com / AP1867.html), and the Protac-binding domain in the chimeric polypeptide of the present invention is FKBP12. F36V (Nabet et al, Nat Chem Biol. 2018 May;14(5):431-441).

[0125] In another embodiment, the small molecule regulatory protein stabilization domain (or controllable destabilization domain) is a CRBN polypeptide substrate domain that can bind to the CRBN protein in response to a drug, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide. Preferably, the CRBN polypeptide substrate domain is a C2H2 zinc finger protein or a fragment thereof (also referred to herein as "zinc finger degron") that can drug-inducibly bind to the CRBN polypeptide. Also provided are cells according to the present invention that contain the chimeric protein according to the present invention, which includes such a small molecule regulatory protein stabilization domain. In such embodiments, the second portion of the chimeric protein according to the present invention includes a small molecule regulatory protein stabilization domain that can interact with and bind to the CRBN protein in the presence of a drug. For example, various IMiDs, including those described herein, have been shown to bind to CRBN proteins, thereby promoting interactions between CRBN proteins and their targets, ubiquitination, and subsequent target protein degradation (see also Buhimschi et al. Biochemistry 2019, 58, 861-864; DOI: 10.1021 / acs.biochem.8b01307 for a recent review of this technology).

[0126] CRBN (Cereblon) is a protein containing 442 amino acid residues that forms the E3 ubiquitin ligase complex with damaged DNA-binding protein 1 (DDB1), culin-4A (CUL4A), and the regulator of culin-1 (ROC1; Angers et al. Nature 443: 590-593). This complex ubiquitinates many other proteins. Thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885 are known to bind to CRBN, respectively (see, for example, Lopez-Girona et al. Leukemia 26: 2326-2335).

[0127] In preferred embodiments, the CRBN polypeptide substrate domain is a C2H2 zinc finger protein or a fragment thereof that can drug-inducibly bind to the CRBN polypeptide. Also provided are cells according to the present invention containing such small-molecule regulatory protein stability domains. Cys2His2-like folding groups (C2H2) are a very common and well-characterized class of zinc fingers in mammalian transcription factors. These domains employ a simple ββα fold, forming two short β strands connected by a short helix following a turn (zinc knuckle; β-turn), and possess an amino acid sequence motif (Pabo et al. Annual Review of Biochemistry (2001). 70: 313-40). X2-Cys-X2,4-Cys-X12-His-X3,4,5-His

[0128] In another preferred embodiment, the chimeric protein comprises a CRBN polypeptide substrate domain containing one or more zinc fingers. Cells according to the present invention are also provided, comprising the chimeric protein according to the present invention, which comprises such a small molecule regulatory protein stability domain.

[0129] Examples include, but are not limited to, specific CRBN polypeptide substrate domains, C2H2 zinc finger proteins or fragments thereof (zinc finger domains) that can bind to CRBN proteins in response to drugs, thereby promoting ubiquitin-mediated degradation of chimeric polypeptides. In preferred embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, ZFN654, ZNF787, ZNF653, ZFP91, ZNF276, ZNF827, or fragments thereof capable of small molecule inductive binding to the CRBN polypeptide, but preferably the fragment is selected from the group consisting of IKZF1 ZF2-3 (SEQ ID NO: 41), IKZF3 ZF2-3 (SEQ ID NO: 42), ZFP91 ZF4-5 (SEQ ID NO: 43), ZNF276 ZF4-5 (SEQ ID NO: 44), ZNF653 ZF4-5 (SEQ ID NO: 45), and ZNF692 ZF4-5 (SEQ ID NO: 46) (see example). Cells according to the present invention containing the chimeric protein according to the present invention, which includes such a small molecule regulatory protein stability domain, are also provided.

[0130] In another preferred embodiment, the CRBN polypeptide substrate domain comprises a complex fusion polypeptide comprising at least a first fragment of a first C2H2 zinc finger protein and a second fragment from a second C2H2 zinc finger protein, wherein the combination of the first and second fragments in the complex fusion polypeptide can be drug-inducibly bound to the CRBN polypeptide. For example, in some embodiments, a β-turn (formed by two short β-strands) from the first C2H2 zinc finger protein may be fused to the α-helix of the second C2H2 zinc finger protein. Cells according to the present invention are also provided, comprising the chimeric protein according to the present invention which includes such a small molecule regulatory protein stability domain.

[0131] The present invention is not particularly limited to specific complex fusion polypeptides that can form or be composed within a CRBN polypeptide substrate domain, but in preferred embodiments, the complex fusion polypeptide comprises a first fragment selected from the β-turns of ZFP91 ZF4 (LQCEICGFTCR; SEQ ID NO: 52), ZFN653 ZF4 (LQCEICGYQCR; SEQ ID NO: 53), ZNF276 ZF4 (LQCEVCGFQCR; SEQ ID NO: 54), and ZNF827 ZF1 (FQCPICGLVIK; SEQ ID NO: 55), and a second fragment selected from the α-helix of IKZF1 ZF2 (QKGNLLRHIKLH; SEQ ID NO: 56), in any conceivable combination. Preferably, the complex fusion polypeptide comprises the β-turn of ZFP91 ZF4 and the α-helix of IKZF1 ZF2, and preferably, the complex fusion polypeptide comprises one selected from SEQ ID NOs: 47-51. Furthermore, cells according to the present invention are also provided, which contain the chimeric protein according to the present invention that includes such a small molecule regulatory protein stability domain.

[0132] According to another embodiment, the CRBN polypeptide substrate-binding domain used in the method of the present invention comprises or further comprises IKZF1 ZF3 (FKCHLCNYACRRRDALTGHLRTH; SEQ ID NO: 57), preferably the CRBN polypeptide substrate-binding domain comprises the β-turn of ZFP91 ZF4, the α-helix of IKZF1 ZF2, and IKZF1 ZF3. Preferably, IKZF1 ZF3 is located at the C-terminus of the second portion of the chimeric protein according to the present invention. Cells according to the present invention comprising such a small molecule regulatory protein stability domain are also provided.

[0133] As will be known to those skilled in the art, in some embodiments, one or more CRBN polypeptide substrate domains capable of binding to CRBN in response to a drug, thereby promoting ubiquitin-mediated degradation of the chimeric protein of the present invention, are incorporated within the chimeric protein of the present invention. The zinc finger degron polypeptide domains (one or more CRBN polypeptide substrate domains capable of binding to CRBN in response to a drug, thereby promoting ubiquitin-mediated degradation of the chimeric protein) are comprised of a single degron polypeptide domain or multiple degron polypeptide domains, and if necessary, the domains of multiple degron polypeptides are linked sequentially or in sequence using a polypeptide linker such as those known in the art.

[0134] As will be known to those skilled in the art, within the CRBN polypeptide substrate-binding domain used in the method of the present invention, different parts (e.g., a β-turn and an α-helix) may be directly adjacent to each other, or they may be linked by a polypeptide linker (including a small sequence of amino acids), such as those known in the art.

[0135] Drugs (e.g., small molecules) suitable for regulating the degradation of chimeric proteins according to the present invention, which include one or more C2H2 zinc finger proteins, fragments, or domains, include so-called immunomodulatory imides (IMiDs), such as thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885 (see, for example, Matyskiela et al. J.Med. Chem. 2018, 61, 2, 535-542; 2017; doi.org / 10.1021 / acs.jmedchem.6b01921 and Gao et al. Biomarker Research (2020) 8:2; doi.org / 10.1186 / s40364-020-0182-y). Those skilled in the art will know how to select a suitable drug, such as a suitable IMiD, to be used in the method according to the present invention.

[0136] Therefore, the cells described in any one of the preceding claims are provided, and the drug that conjugates the CRBN polypeptide substrate domain to the CRBN protein, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide, is an IMiD selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885.

[0137] Therefore, a cell according to any one of the preceding claims is provided, further comprising a drug that conjugates a CRBN polypeptide substrate domain to a CRBN protein, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide, wherein the drug is preferably an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885, and preferably the drug conjugates to the CRBN polypeptide substrate domain.

[0138] As those skilled in the art will know, the chimeric polypeptides of the present invention can exist in different forms within the cells of the present invention. For example, if the small molecule regulatory protein stability domain (or controllable destabilization domain) is SED, the chimeric polypeptides can exist in the cells with or without the degron sequence, depending on the presence of the homologous small compound / protease inhibitor.

[0139] Furthermore, the present invention provides cells in which the ITAM is an ITAM contained in a T cell receptor (TCR) complex and / or a chimeric antigen receptor (CAR) and / or NKR complex, preferably the CD3 ζ chain, CD3 ε chain, CD3 δ chain, CD3 γ chain, the γ chain of the immunoglobulin receptor FcεRI, and an ITAM contained in DAP12.

[0140] As explained, the SH2 domain may originate from a protein that binds a phosphorylated immune receptor tyrosine-based activation motif (ITAM).

[0141] ITAMs are found within the intracellular domains of cell signaling molecules such as the CD3 zeta, epsilon, gamma, and delta chains of the T cell receptor complex, as well as certain Fc receptors (Love et al, Cold Spring Harb Perspect Biol, 2010 Jun; 2(6): a002485).

[0142] In NK cells, specific activated NK cell receptors (NKRs) form complexes with signaling molecules containing ITAMs, such as the CD3 ζ chain, the γ (gamma) chain of the immunoglobulin receptor FcεRI, and DAP12. For example, NK cell receptors (NKRs) NKp46 and NKp30 associate with the γ (gamma) chain of the immunoglobulin receptor FcεRI and the CD3 ζ chain, while NKp44 associates with the signaling adapter DAP12 (Barrow et al, Front Immunol. 2019;10:909). Therefore, in one embodiment of the present invention, the cells according to the present invention are NK cells.

[0143] Domains containing ITAMs are also used in the design of chimeric antigen receptors (CARs). The CD3 ζ chain contains three ITAMs, while the CD3 ε chain, the γ (gamma) chain of the immunoglobulin receptor FcεRI, and the DAP12 signaling domain contain one ITAM. These are used in the design of various CARs (Ren-Heidenreich et al, Cancer Immunol Immunother. 2002 Oct;51(8):417-23, Nolan et al, Clin Cancer Res. 1999 Dec;5(12):3928-41, Topfer et al, J Immunol. 2015 Apr 1;194(7):3201-12).

[0144] Two tyrosine residues within ITAM are phosphorylated by Src kinase family members such as Lck. Phosphorylated ITAM then functions as a platform for docking with the SH2 domains of Zap70 and Syk (Long et al, Annu Rev Immunol. 2013; 31: 10.1146 / annurev-immunol-020711-075005).

[0145] The semi-ITAM symbol is easily recognizable as tyrosine isolated from leucine or isoleucine with any two other amino acids, giving the symbol YxxL / l. Two of these symbols are separated by 6-8 amino acids, forming the common ITAM sequence YxxL / lx(6-8)YxxL / l. In a preferred embodiment, the ITAM-containing domain may be a CD3 ζ-chain domain or may contain a CD3 ζ-chain domain. In another preferred embodiment, the ITAM-containing domain may be a CD3 ε-chain domain or may contain a CD3 ε-chain domain. Furthermore, in yet another preferred embodiment, the ITAM-containing domain may be the γ (gamma) chain of the immunoglobulin receptor FcεRI or may contain this γ-chain. Furthermore, in yet another preferred embodiment, the ITAM-containing domain may be a DAP12 domain or may contain a DAP12 domain.

[0146] The present invention also provides cells that further comprise T cell receptors (T cell receptor complexes) and / or chimeric antigen receptors (CARs) or NK cell receptors (NK cell receptor complexes). Preferably, the cells are T cells, CAR T cells, NK cells and / or CAR NK cells.

[0147] Preferably, the cells are T cells expressing a TCR complex and / or a CAR complex. Preferably, the TCR complex or CAR complex comprises a CD3 ζ-chain domain containing an ITAM, or any other domain having an ITAM as disclosed herein.

[0148] Preferably, the cells are NK cells expressing an NKR complex and / or a CAR complex. Preferably, the NKR complex or CAR complex comprises a CD3 ζ-chain domain containing an ITAM, or any other domain having an ITAM as disclosed herein.

[0149] Those skilled in the art are well familiar with T cells and / or CAR T cells. T cells, or T lymphocytes, play a central role in cell-mediated immunity. They are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface.

[0150] T cells include various types such as T helper cells (TH cells), cytolytic T cells, and regulatory T cells. TH cells express CD4 on their surface and are activated when peptide antigens are presented on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subspecies that secrete different cytokines to promote different types of immune responses.

[0151] Hemolytic T cells (TC cells, CTLs) destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize targets by binding to MHC class I-related antigens present on the surface of all nucleated cells.

[0152] Regulatory T cells (Tregs) are cells that suppress immune responses by secreting molecules such as IL-10, and are characterized by the expression of the transcription factor FOXP3.

[0153] Memory T cells are a subset of antigen-specific T cells that persist long after an infection has healed. When exposed to the antigen again, they rapidly expand into a large number of effector T cells, thus giving the immune system a "memory" of past infections. Memory cells are either CD4+ or CD8+.

[0154] Preferably, the T cells are CD4-positive T cells. Preferably, the T cells are CD8-positive T cells. CAR T cells are T cells that express the CAR complex.

[0155] Natural killer cells (or NK cells) are a type of cell-degrading cell that is part of the innate immune system. NK cells respond to natural signals from virus-infected cells in a peptide-independent manner.

[0156] NK cells are defined as large granular lymphocytes and are a third type of cell differentiated from common lymphoid progenitor cells that produce B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus.

[0157] The cells of the present invention may be any type of cell, in particular T cells, CAR T cells, NK cells, or CAR NK cells.

[0158] T cells (including CAR T cells) or NK cells (including CAR NK cells) expressing the molecule of the present invention can be created in vitro, for example, from the patient's own peripheral blood or from donor peripheral blood.

[0159] Furthermore, the present invention also provides cells derived from a protein whose SH2 domain is selected from the group consisting of Zap70, Syk, and Lck.

[0160] In particular, the chimeric polypeptide according to the present invention, comprising a first portion containing Zap70, Syk, and SH2 domains derived from Lck, was found to be suitable according to the present invention.

[0161] ZAP70 is a protein that is normally expressed near the cell membrane of T cells and natural killer cells and plays an important role in T cell signaling. It has a molecular weight of 70 kDa and consists of two N-terminal SH2 domains and a C-terminal kinase domain. It is a member of the protein tyrosine kinase family. The human ZAP70 protein has UniProtKB acceptance number P43403. This sequence is 619 amino acids long and is shown as SEQ ID NO: 37.

[0162] Syk is expressed in thymocytes, intraepithelial γδ T cells, naive αβ T cells, and B cells (Latour et al, Mol Cell Biol. 1997 Aug; 17(8): 4434-4441). Syk has high homology to ZAP70 and has the same domain structure with two N-terminal SH2 domains and a C-terminal kinase domain. In B cells, Syk deficiency can be reconstituted by Zap70 (Kong et al, Immunity. 1995 May; 2(5): 485-92). Similarly, ZAP70 deficiency can be reconstituted by Syk in T cells (Williams et al, Mol Cell Biol. 1998 Mar; 18(3): 1388-99). The human Syk protein has UniProtKB acceptance number P43405. This sequence is 635 amino acids long and is shown as Sequence ID No. 38.

[0163] Lck (also known as p56-LCK) is expressed in lymphocytes. Lck plays a crucial role in the TCR signaling pathway. In T cells, it constitutively binds to the cytoplasmic domains of the CD4 and CD8 coreceptors. When the TCR is activated by the peptide-MHC complex, Lck approaches the TCR complex, and the ITAM residue of the CD3 subunit is phosphorylated by Lck. The phosphorylated ITAM functions as a docking site for the SH2 domain of Zap70 (Simeoni, Oncotarget. 2017 Nov 28; 8(61): 102761-102762). The domain structure of Lck is an SH4-specific domain (UD)-SH3-SH2-kinase domain. The SH2 domain is required for interaction with phosphorylated ITAM, and SH4 is required for membrane association (Ngoenkam et al, Immunology. 2018 Jan; 153(1): 42-50). The human Lck protein has UniProtKB acceptance number P06239. This sequence is 509 amino acids long and is represented by SEQ ID NO: 39. Preferably, Zap70, Syk, and Lck are human Zap70, Syk, and Lck.

[0164] The present invention also provides cells in which the chimeric polypeptide contains two or more protein-derived SH2 domains that bind to an activation motif based on phosphorylated immune receptor tyrosine.

[0165] According to the present invention, the presence of one SH2 domain in the chimeric polypeptide is preferred, but in some embodiments, there may be two or more SH2 domains capable of binding to phosphorylated ITAM. Preferably, phosphorylated ITAM present in a T cell receptor (complex), CAR (complex), or NKR (complex) may constitute the first portion of the chimeric polypeptide (as described elsewhere herein, the first portion of the chimeric polypeptide means the portion containing an SH2 domain from a protein that binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM)). As disclosed herein, this first portion may be located anywhere in the chimeric polypeptide according to the present invention, and “first portion” does not necessarily mean “at the N-terminus.” The same applies to the second and third portions of the chimeric polypeptide as defined and disclosed herein and further discussed herein. The two or more SH2 domains may be from the same (natural) protein or from two or more different proteins.

[0166] As those skilled in the art will know, for example, after the SH2 domain, other domains are intended to constitute the first part of the chimeric polypeptide, such as those present in the chimeric polypeptide tested in the examples.

[0167] The present invention also provides cells in which ITIM and / or ITSM are derived from an inhibitory receptor protein, preferably an inhibitory immune receptor protein, preferably a protein selected from the group consisting of PD1, BTLA, SIRPα, Siglec 5, Siglec 9, Siglec 11, PECAM1, or LY9. Preferably, the inhibitory receptor protein, inhibitory immune receptor protein, or protein selected from the group consisting of PD1, BTLA, SIRPα, Siglec 5, Siglec 9, Siglec 11, PECAM1, or LY9 is a human-derived protein.

[0168] PD1 (also known as PD-1) is encoded by the PDCD1 gene. PD1 is a type I transmembrane protein. Through interaction with its ligands PD-L1 / PD-L2, it suppresses the effector function of cytotoxic T cells. The UniProtKB acceptance number for human PD1 is Q15116. This sequence is 288 amino acids long. The cytoplasmic domain of PD1 contains ITIM and ITSM motifs. Phosphorylated ITSM of the cytoplasmic domain of PD1 recruits SHP-2 phosphatase, which dephosphorylates key signaling molecules in the TCR signaling pathway, such as ZAP70, PKCtheta, and CD3 zeta (CD247), leading to suppression of TCR signaling (Bardhan et al, Front Immunol. 2016; 7: 550) and CD28-mediated co-stimulation (Hui et al, Science. 2017 Mar 31; 355(6332): 1428-1433). For example, a suitable third moiety used in the chimeric polypeptide according to the present invention, which preferably includes an immune receptor tyrosine-based switch motif (ITSM), and preferably an immune receptor tyrosine-based inhibitor motif (ITIM), is characterized by SEQ ID NO: 17 (representing the cytoplasmic domain of PD1).

[0169] B lymphocyte and T lymphocyte attenuation factor (BTLA) is primarily expressed on T cells, B cells, and mature lymphocytes (Yue et al, Front Immunol. 2019; 10: 617). It is an immunoregulatory receptor that plays a crucial role in immune tolerance. Like PD1, BTLA is a type I transmembrane glycoprotein. BTLA receptor involvement induces SHP-1 / SHP-2 recruitment and suppression of IL-2 secretion in T cells (Watanabe et al, Nat Immunol. 2003 Jul;4(7):670-9). The human BTLA protein has UniProtKB acceptance number Q7Z6A9. This sequence is 289 amino acids long. The cytoplasmic domain of BTLA contains ITIM and ITSM motifs.

[0170] For example, a suitable third moiety used in the chimeric polypeptide according to the present invention, preferably comprising an immune receptor tyrosine-based switch motif (ITSM), and preferably an immune receptor tyrosine-based inhibitor motif (ITIM), is characterized by SEQ ID NO: 15 (representing the cytoplasmic domain of BTLA).

[0171] SIRPA (also known as SIRPα, BIT, MFR, MYD1, PTPNS1, SHPS1, SIRP) is expressed in myeloid cells. SIRPA negatively regulates phagocytosis, mast cell activation, and dendritic cell activation through binding to ligand CD47 (Timms et al, Curr Biol. 1999 Aug 26;9(16):927-30, Latour et al, J Immunol. 2001 Sep 1;167(5):2547-54, Matlung et al, Immunol Rev. 2017 Mar;276(1):145-164. doi: 10.1111 / imr.12527). In macrophages, SIRPA primarily associates with SHP-1 (Veillette et al, J Biol Chem. 1998 Aug 28;273(35):22719-28). SIRPA is a type I transmembrane protein. The human SIRPA protein has UniProtKB acceptance number P78324. This sequence is 504 amino acids long. The cytoplasmic domain of SIRPA contains two ITIM and one ITSM motif.

[0172] For example, a suitable third moiety that may be used in the chimeric polypeptide according to the present invention, preferably comprising an immune receptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immune receptor tyrosine-based inhibitor motif (ITIM), is characterized by SEQ ID NO: 19 (representing the cytoplasmic domain of SIRPa).

[0173] PECAM1 (also known as PECAM-1 or CD31) is expressed in T cells, B cells, platelets, monocytes, macrophages, and neutrophils (Newton-Nash et al, J Immunol. 1999 Jul 15;163(2):682-8). PECAM1 inhibits T cell and B cell signaling through the recruitment of SHIP1, SHP-1, and SHP-2 (Marelli-Berg et al. J Cell Sci. 2013 Jun 1;126(Pt 11):2343-52). In macrophages, ligand binding to PECAM1 results in the recruitment of SHP-1 and SHP2, suppression of TNF-α, IL-6, and IFN-β production, and TLR4 signaling (Rui et al, J Immunol. 2007 Dec 1;179(11):7344-51). PECAM1 negatively regulates platelet signaling pathways (Jones et al, FEBS Lett. 2009 Nov 19;583(22):3618-24). PECAM1 is a type I transmembrane protein. The human PECAM1 protein has UniProtKB acceptance number P16284. This sequence is 738 amino acids long. The cytoplasmic domain of PECAM1 contains ITIM and ITSM motifs. For example, a preferred third moiety used in the chimeric polypeptide according to the present invention, which includes an immunoreceptor tyrosine-based switch motif (ITSM), preferably ITSM and an immunoreceptor tyrosine-based inhibitor motif (ITIM), is characterized by SEQ ID NO: 18 (representing the cytoplasmic domain of PECAM1).

[0174] Sialic acid-binding immunoglobulin lectins (SIGLECs) are a group of immunoregulatory receptors primarily expressed in hematopoietic cells (Bornhofft et al, Dev Comp Immunol. 2018 Sep;86:219-231). SIGLEC 5 (also known as CD33L2 or OBBP2) is expressed in monocytes, neutrophils, and B cells; SIGLEC 9 is expressed in neutrophils, monocytes, dendritic cells, and NK cells; and SIGLEC 11 is expressed in macrophages (Macauley et al, Nat Rev Immunol. 2014 Oct; 14(10): 653-666). Most siglecs possess inhibitory ITIM / ITSM motifs, recruiting SHP1 and SHP2 and acting as negative regulators of the immune system (Crocker et al, Nat Rev Immunol. 2007 Apr;7(4):255-66, Avril et al, J Biol Chem. 2005 May 20;280(20):19843-51, Haas et al, Cancer Immunol Res. 2019 May;7(5):707-718, Angata et al, J Biol Chem. 2002 Jul 5;277(27):24466-74). Siglec 5, siglec 9, and siglec 11 are type I transmembrane proteins. They possess ITIM and ITSM motifs in their cytoplasmic domains. The human siglec 5 protein has UniProtKB acceptance number O15389. This sequence is 551 amino acids long. The human siglec 9 protein has UniProtKB acceptance number Q9Y336. This sequence is 463 amino acids long. The human siglec 11 protein has UniProtKB acceptance number Q96RL6. This sequence is 698 amino acids long. For example, a suitable third moiety used in the chimeric polypeptide according to the present invention, which includes an immune receptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immune receptor tyrosine-based inhibitor motif (ITIM), is characterized by SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 20 (representing the cytoplasmic domains of siglecs 5, 9, and 11, respectively).

[0175] The T lymphocyte surface antigen Ly-9 (also known as LY9, SLAMF3, and CD229) is expressed on thymocytes, mature T and B lymphocytes (de la Fuente et al, Blood. 2001 Jun 1;97(11):3513-20). It interacts with SHIP-1 and SHP-2 (Punet-Ortiz et al, Front Immunol. 2018 Nov 16;9:2661) and has been reported to function as a negative regulator of immune responses, contributing to peripheral cell tolerance (de Salort et al, Front Immunol. 2013; 4: 225). LY9 is a type I transmembrane protein. The human LY9 protein has UniProtKB acceptance number Q9HBG7. This sequence is 655 amino acids long. The cytoplasmic domain of LY9 contains two ITSM motifs. For example, a suitable third moiety used in the chimeric polypeptide according to the present invention, which preferably includes an immune receptor tyrosine-based switch motif (ITSM), and preferably an immune receptor tyrosine-based inhibitor motif (ITIM), is characterized by SEQ ID NO: 16 (representing the cytoplasmic tail of LY9).

[0176] In some embodiments, ITSM and ITIM are obtained from or derived from the same inhibitory protein, while in other embodiments, ITSM and ITIM are derived from different inhibitory proteins.

[0177] Furthermore, cells according to the present invention are also provided, wherein the chimeric polypeptide comprises ITSM and ITIM.

[0178] It was unexpectedly discovered that the presence of both ITSM and ITIM in the third portion of the chimeric polypeptide of the present invention is particularly advantageous (see Examples).

[0179] The present invention also provides cells in which, preferably, the first portion is located at the N-terminus of the chimeric polypeptide, the second portion is located at the C-terminus of the chimeric polypeptide, and preferably, the third portion is located between the first and second portions.

[0180] Furthermore, as disclosed elsewhere in this Specification, the first and third parts may be directly adjacent to each other or separated by an additional sequence of amino acids. Also, as disclosed elsewhere in this Specification, the second and third parts may be directly adjacent to each other or separated by an additional sequence of amino acids. While not limited to any particular order, this specific order of the first, second, and third parts has been found to be advantageous.

[0181] Also disclosed are cells according to the present invention that further comprise a protease inhibitor bound to an inhibitory protease, or a Protac bound to a proteolytic target chimeric (Protac) binding domain, or a drug that binds a CRBN polypeptide substrate domain to a CRBN protein. This promotes ubiquitin pathway-mediated degradation of the chimeric polypeptide, and preferably the drug is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885, as disclosed and described elsewhere herein. In preferred embodiments, the protease inhibitor is a protease inhibitor as disclosed herein. In preferred embodiments, the Protac is a Protac as disclosed herein. In preferred embodiments, the CRBN polypeptide substrate domain, which can bind to the CRBN protein in response to a drug and thereby promote ubiquitin-mediated degradation of the chimeric polypeptide, is as disclosed herein, and preferably the drug is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885.

[0182] Furthermore, the following cells according to the present invention are also disclosed. a) The SH2 domain contains or consists of an amino acid sequence having at least 80% identity with the amino acid sequences according to SEQ ID NOs: 7-11, or contains or consists of an amino acid sequence having at least 80% identity with the amino acid sequences according to SEQ ID NOs: 12-14. b) The first portion of the chimeric polypeptide contains or consists of an amino acid sequence having at least 80% identity with the amino acid sequence according to SEQ ID NOs. 7-11, or contains or consists of an amino acid sequence having at least 80% identity with the amino acid sequence according to SEQ ID NOs. 12-14. c) ITAM is YxxL / lx(6-8)YxxL / l d) The SH2 domain is an SH2 domain that binds to phosphorylated ITAM composed of SEQ ID NOs: 1-6, or an SH2 domain that binds to an amino acid sequence that has 80% or more identity with the amino acid sequence following SEQ ID NOs: 1-6. e) ITIM is S / I / V / LxYxxI / V / L and / or ITSM is TxYxxV / I, f) ITSM and / or ITIM consist of sequence numbers 15-22. g) The third portion of the chimeric polypeptide contains or consists of an amino acid sequence that has at least 80% identity with the amino acid sequence according to SEQ ID NOs. 15-22. h) The second portion of the chimeric polypeptide contains or consists of an amino acid second sequence according to SEQ ID NOs. 35 or 40 or SEQ ID NOs. 41-57, and / or i) The chimeric polypeptide contains an amino acid sequence that has 80% or more identity with the amino acid sequence according to SEQ ID NOs. 23-34, SEQ ID NOs. 36, or SEQ ID NOs. 58-68.

[0183] Those skilled in the art will know that any defined amino acid sequence associated with a first portion of a chimeric polypeptide can be combined with any defined amino acid sequence associated with a third portion of the chimeric polypeptide and / or a second portion of the chimeric polypeptide. In other words, any first portion, any second portion, and any third portion as disclosed herein can be combined to form the first, second, and third portions of the chimeric polypeptide according to the present invention, respectively. In preferred embodiments, the first portion of the chimeric polypeptide comprises an amino acid sequence as defined in a), b), c), and / or d), and the third portion of the chimeric polypeptide comprises an amino acid sequence as defined in e), f), or g). As can be seen from this disclosure, different first portions of a chimeric protein can be combined with different third portions of a chimeric polypeptide.

[0184] Furthermore, those skilled in the art will know that, in addition to the amino acid sequences of the first, second, and third portions of the chimeric protein as defined herein, the chimeric polypeptide according to the present invention may include further portions. That is, the chimeric polypeptide disclosed herein is not limited to chimeric polypeptides consisting only of the first, second, or first, second, and third portions as defined herein. The chimeric polypeptide according to the present invention may include additional (extended) or additional (functional) portions of amino acids.

[0185] Therefore, in a preferred embodiment, the SH2 domain, such as that present in the first portion of the chimeric polypeptide, may contain or consist of amino acid sequences having at least 80% identity to the amino acid sequences according to SEQ ID NOs: 7-11, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, or may contain or consist of amino acid sequences having at least 80% identity to the amino acid sequences according to SEQ ID NOs: 12-14, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity. As those skilled in the art will know, these sequences defined above also include sequences in which 1, 2, 3, 4, 5, 6, or 10 amino acids are deleted, substituted, or inserted.

[0186] Therefore, in a preferred embodiment, the first portion of the chimeric polypeptide may include or consist of amino acid sequences having at least 80% identity, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to the amino acid sequences according to SEQ ID NOs. 12-14, or it may include or consist of amino acid sequences having at least 80% identity, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity. As those skilled in the art will know, sequences in which 1, 2, 3, 4, 5, 6, or 10 amino acids are deleted, substituted, or inserted from the sequences defined above are also included. The first portion also includes the addition (extension) of amino acids adjacent to the above-defined amino acids, insofar as the first portion of the chimeric polypeptide is functional in the context of the present invention.

[0187] Therefore, when ITAM is phosphorylated, the ITAM to which the SH2 domain of the first portion of the chimeric polypeptide binds is YxxL / lx(6-8)YxxL / l, as explained above.

[0188] Therefore, in a preferred embodiment, the SH2 domain present in the first portion of the chimeric polypeptide may bind to a phosphorylated ITAM composed of SEQ ID NOs: 1-6, or to an amino acid sequence that has at least 80%, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the amino acid sequence according to SEQ ID NOs: 1-6.

[0189] Therefore, the third portion of the chimeric polypeptide comprising ITSM, preferably ITSM and ITIM, may comprise ITSM and / or ITIM, wherein ITIM is S / I / V / LxYxxI / V / L and / or ITSM is TxYxxV / I.

[0190] Therefore, the ITSM and / or ITIM comprising the third part of the chimeric polypeptide may be the ITSM and / or ITIM comprising SEQ ID NOs. 15-22.

[0191] Therefore, the third portion of the chimeric polypeptide may include or consist of an amino acid sequence having at least 80%, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the amino acid sequence according to SEQ ID NOs. As those skilled in the art will know, this also includes sequences defined above in which 1, 2, 3, 4, 5, 6, or 10 amino acids are deleted, substituted, or inserted. The third portion may also include the addition (extension) of amino acids adjacent to the above-defined amino acids, insofar as the third portion of the chimeric polypeptide is functional in the context of the present invention.

[0192] Accordingly, the second portion of the chimeric polypeptide according to the present invention may include or consist of an amino acid sequence according to SEQ ID NO: 35, SEQ ID NO: 40, or SEQ ID NOs: 41-57. As those skilled in the art will know, this also includes sequences defined above in which 1, 2, 3, 4, 5, 6, or 10 amino acids are deleted, substituted, or inserted. The third portion may also include the addition (extension) of amino acids adjacent to the above-defined amino acids, insofar as the third portion of the chimeric polypeptide is functional in the context of the present invention. Furthermore, an amino acid sequence having at least 80% identity, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to the amino acid sequence according to SEQ ID NO: 35, SEQ ID NO: 40, or SEQ ID NOs: 41-57 is intended.

[0193] Therefore, a chimeric polypeptide comprises or consists of an amino acid sequence having at least 80%, preferably at least 81, 83, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with an amino acid sequence according to SEQ ID NOs. As those skilled in the art will know, this also includes sequences defined above with 1, 2, 3, 4, 5, 6, 10, or 15 amino acids deleted, substituted, or inserted. A chimeric polypeptide may also include the addition (extension) of amino acids adjacent to the defined amino acids, insofar as the chimeric polypeptide according to the present invention is functional in the context of the present invention.

[0194] In another aspect of the present invention, chimeric proteins as defined herein are also provided. Chimeric proteins may be present in cells or in any other form.

[0195] According to another aspect of the present invention, nucleic acids comprising polynucleotides encoding a chimeric polypeptide according to the present invention are also provided.

[0196] Naturally, the nucleic acids according to the present invention are preferably introduced into cells, such as T cells, CAR T cells, or NK cells, and expressed in such cells, thereby expressing the chimeric polypeptide according to the present invention in such cells. The nucleic acids are introduced in the form of a vector or plasmid. Depending on the embodiment, the nucleic acids of the present invention are incorporated into the genome of cells such as T cells, CAR T cells, or NK cells. Such T cells are used, for example, to introduce modified T cell receptors and / or CARs.

[0197] In some embodiments, the nucleic acid of the present invention is introduced into T cells or NK cells before, after, or simultaneously with the nucleic acid encoding a (modified) T cell receptor or CAR. The nucleic acid of the present invention and the nucleic acid encoding a (modified) T cell receptor or CAR may be introduced using two separate vectors, or using a single vector containing both nucleic acids. Similarly, in some embodiments, the nucleic acid of the present invention is introduced into NK cells before, after, or simultaneously with the nucleic acid encoding a (modified) NK cell receptor or CAR. The nucleic acid of the present invention and the nucleic acid encoding a (modified) NK cell receptor or CAR may be introduced using two separate vectors, or using a single vector containing both nucleic acids. Therefore, a vector containing nucleic acid according to the present invention is also provided. Furthermore, cells containing nucleic acids according to the present invention are also provided.

[0198] Furthermore, pharmaceutical compositions comprising cells or nucleic acids according to the present invention are also provided. Preferably, the cells are T cells, CAR T cells, NK cells, or CAR NK cells. The T cells may be derived from the patient being treated or may be allogeneic.

[0199] Immunotherapy, which involves transplanting self-antigen-specific T cells produced in vitro, is expected to be a promising treatment for viral infections and cancer. T cells used in immunotherapy are generated through the proliferation of antigen-specific T cells or by rearranging T cells through genetic engineering. Successful methods have been developed to create antigen-specific T cells for immunotherapy by introducing genes for T cell receptors and chimeric antigen receptors (CARs).

[0200] The present invention has shown that it is possible to provide T cells, CAR T cells, NK cells, or CAR NK cells to patients who currently require immunotherapy or immunotherapy (e.g., treatment with T cells, CAR T cells, NK cells, and CAR NK cells), and to modulate the T cell or NK cell function of these cells (e.g., cytotoxic activity and / or cytokine secretion of T cells or NK cells) as disclosed herein. Those skilled in the art are well familiar with various methods of immunotherapy using chimeric antigen receptor gene-modified T cells or (recombinant) T cell receptors, as outlined, for example, by Rosenberg et al, Science. 2015 Apr 3;348(6230):62-8 and June et al, Science. 2018 Mar 23;359(6382):1361-1365, and unmodified or CAR gene-modified NK cells, as outlined by Hu et al, Front Immunol. 2019; 10: 1205, Kloess et al, Transfus Med Hemother. 2019 Feb; 46(1): 4-13 and Suen et al, Cancer Invest. 2018;36(8):431-457.

[0201] The cells, chimeric polypeptides, and / or nucleic acids according to the present invention are suitably used in such immunotherapy, as those skilled in the art will understand based on this disclosure. Therefore, cells according to the present invention that can be used as pharmaceuticals are also provided.

[0202] Therefore, cells according to the present invention are also provided for use in treating target cancers. Cancers suitable for treatment are selected from the group consisting of hematological cancers, particularly B-cell carcinoma, melanoma, breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, and prostate cancer.

[0203] Preferably, the cells used as pharmaceuticals or for the treatment of cancer are T cells or CAR T cells, or NK cells or CAR NK cells.

[0204] Furthermore, cells used as pharmaceuticals, preferably cells used for the treatment of the target cancer according to the present invention, are also provided, and this treatment is a) To administer to a population of cells according to the present invention, b) If necessary, administer an inhibitor of an inhibitory protease, Protac which binds to the proteolytic target chimeric (Protac) binding domain, or a drug which binds the CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885) to the target, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide. c) If step b) is performed as necessary, the step includes increasing or decreasing the concentration of an inhibitor of an inhibitory protease that binds the CRBN polypeptide substrate domain to the CRBN protein, Protac, or a drug, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide, wherein the drug is preferably an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885.

[0205] As disclosed elsewhere in this specification, the T cell function or NK cell function of cells according to the present invention can be controlled by the expression of the chimeric polypeptide according to the present invention. In the absence of inhibitory protease inhibitors (when the second portion of the chimeric polypeptide includes SED), or in the absence of Protac that binds to the Protac-binding domain of the second portion of the chimeric polypeptide according to the present invention, or in the absence of a drug that binds the CRBN polypeptide substrate domain to the CRBN protein and thereby promotes ubiquitin-mediated degradation of the chimeric polypeptide (preferably the drug is IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885), T cell function is suppressed by inhibition of TCR or CAR signaling by the chimeric polypeptide according to the present invention, or NK function is suppressed by inhibition of NKR or CAR signaling by the chimeric polypeptide according to the present invention. In the presence of an inhibitor or such Protac or such agent (preferably an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885) that conjugates the CRBN polypeptide substrate domain to the CRBN protein and thereby promotes ubiquitin-mediated degradation of the chimeric polypeptide, the chimeric polypeptide according to the present invention is degraded, leading to the release of suppression of T cell or NK cell function (cytokine release / cytotoxicity). Since the modulation of T cell function or NK function is reversible and dose-dependent, T cell function or NK function can be regulated as part of the treatment of the target.

[0206] Furthermore, a method for providing cells according to the present invention is also provided, which includes contacting cells with nucleic acids according to the present invention, preferably including the step of contacting the cells with nucleic acids in vitro.

[0207] Furthermore, a method for controlling the expression of chimeric polypeptides in cells is also provided. This method involves contacting cells expressing the chimeric polypeptide according to the present invention with an inhibitor of an inhibitory protease, a Protac that binds to a proteolytic target chimeric (Protac) binding domain, or a drug that binds a CRBN polypeptide substrate domain to a CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide. Preferably, the drug is an IMiD, and is preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885. Preferably, cells are brought into contact with an inhibitory protease inhibitor or Protac, or a drug that conjugates the CRBN polypeptide substrate domain to the CRBN protein, either in vitro or preferably in vivo, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide. Preferably, the drug is an IMiD, and is preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885.

[0208] The expression of the chimeric polypeptide according to the present invention is controlled by adjusting its level, i.e., concentration, by degrading or inducing degradation of the chimeric polypeptide according to the present invention, as disclosed elsewhere in this specification.

[0209] Furthermore, methods for controlling the cytotoxic activity of T cells and / or NK cells, and / or methods for controlling cytokine secretion by T cells and / or NK cells are also provided. a) A step of expressing a chimeric polypeptide according to the present invention in T cells and / or NK cells, b) Contacting T cells and / or NK cells with an inhibitor of an inhibitory protease, Protac which binds to a proteolytic target chimeric (Protac) binding domain, or a drug which binds a CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885), thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide, and c) If necessary, increasing or decreasing the concentration of an inhibitory protease inhibitor or Protac, or a drug that conjugates the CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885), thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide. Includes.

[0210] Those skilled in the art are well aware of the cytotoxic activity of T cells and NK cells and / or methods for determining cytokine secretion by such T cells, e.g., IFNγ, IL-2, TNFα, IL-10, IL-4, and NK cells, e.g., IFNγ, IL-2, TNFα. The T cells and / or NK cells may be derived from the patient being treated or from an allogeneic lineage. A method for treating cancer in the subject is also provided. This method is a) A step of providing cells according to the present invention, b) Optionally, administer an inhibitor of an inhibitory protease, Protac which binds to the proteolytic target chimeric (Protac) binding domain, or a drug which binds the CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885) to the target, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide. c) If necessary, when step b) is performed, the step includes increasing or decreasing the concentration of an inhibitory protease inhibitor or Protac, or a drug that conjugates the CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885), thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide. The T cells and / or NK cells may be derived from the patient being treated or allogeneic cells.

[0211] Furthermore, methods for controlling the cytotoxic activity of target T cells and / or NK cells, and / or methods for controlling cytokine secretion by target T cells and / or NK cells are also provided. This method is a) A step of providing cells according to the present invention to a target, b) Administering an inhibitory protease inhibitor, a Protac that binds to a proteolytic target chimeric (Protac) binding domain, or a drug that binds a CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is an IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885) to the target, thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide, and c) The process includes, if necessary, increasing or decreasing the concentration of an inhibitory protease inhibitor or Protac, or a drug that conjugates the CRBN polypeptide substrate domain to the CRBN protein (preferably the drug is IMiD, preferably selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iverdamide), and CC-885), thereby promoting ubiquitin-mediated degradation of the chimeric polypeptide. T cells and / or NK cells may be derived from the patient being treated or allogeneic cells.

[0212] Naturally, all details, embodiments, and priority of choices considered in relation to one aspect of the embodiment of the present invention also apply to other aspects or embodiments of the present invention, and therefore it is not necessary to separately detail such details, embodiments, and priority of choices in relation to all aspects.

[0213] While the present invention has been described in general terms, the same will be better understood by referring to the following embodiments, which are provided for illustrative purposes and are not intended to limit the invention. Further aspects and embodiments will be apparent to those skilled in the art. [Examples]

[0214] Overview To develop a system that can reversibly control the activity of T cells expressing any antigen receptor, we devised a method to transfer inhibitory signals to CAR or TCR signaling complexes in a dose-configurable manner. A common characteristic of CAR and TCR antigen receptors is that signaling mediated by these receptors leads to phosphorylation of the immune receptor tyrosine activation motif (ITAM). When the antigen receptor binds to an agonist ligand, it activates Lck, which phosphorylates the ITAM of the CD3 complex. Once the CD3 ζ chain is phosphorylated, the Zap70 protein is recruited to the phosphorylated ITAM of the CD3 ζ chain via its SH2 domain.

[0215] We hypothesized that if a signaling domain in an inhibitory receptor fused with an SH2 domain that interacts with ITAMs, such as the Zap70 SH2 domain, or with an SH2 domain of another protein that binds to phosphorylated immune receptor tyrosine activation motifs (ITAMs) like Syk or Lck, and then migrated to the activating antigen receptor, TCR / CAR signaling might be suppressed.

[0216] When bound to natural ligands, various inhibitory receptors such as PD1, BTLA, SIRPα, Siglec 5, Siglec 9, Siglec 11, PECAM1, and LY9 have been shown to interfere with the activation of immune cells.

[0217] To support the concept of inhibitory domain delivery by the SH2 domain, we initially focused on the intracellular domain of PD1 because signaling related to cytokine production and cytotoxicity is reversible (Barber et al, Nature. 2006 Feb 9;439(7077):682-7), and it has been shown that the physical proximity of PD1 to the TCR microcluster is important for T cell suppression (Yokosuka et al, J Exp Med. 2012 Jun 4;209(6):1201-17). Based on these observations, we created a Zap70 2xSH2 domain-PD1 tail fusion protein (hereinafter abbreviated as Zap70-PD1) with the aim of bringing the repressive domain of PD1 closer to the TCR complex (Figure 1A-B). The Zap70 2xSH2 domain binds to the phosphorylated CD3 ζ chain and initiates the TCR signaling pathway by phosphorylating downstream targets (Wang et al, Cold Spring Harb Perspect Biol, 2010 May;2(5):a002279).

[0218] To evaluate the effect of Zap70-PD1 on T cell activation, primary human T cells were transformed with a high-affinity CDK4 nascent antigen-specific class I restriction TCR (Stronen et al, Science. 2016 Jun 10;352(6291):1337-41), and with either a Zap70-PD1 fusion, a Zap70 SH2 domain without an inhibitory tail (Zap70 2xSH2), an intracellular PD1 domain (PD1 tail), or a vector control.

[0219] Analysis of T cell cytokine production (IFNγ, IL2, TNFα) or T cell degranulation (LAMP-1 cell surface expression) in T2 tumor cells treated with CDK4 neoantigen under constant temperature conditions revealed that expression of the free PD1 tail did not significantly alter T cell function. Expression of the Zap70 2xSH2 domain without the PD1 tail slightly inhibited T cell function (Figure 1C-D), which may be explained by competition with endogenous wild-type Zap70.

[0220] In particular, linking the intracellular domain of PD1 to the SH2 domain of Zap70 was observed to highly efficiently suppress both T cell cytokine production and T cell degranulation (the proportion of reactive cells decreased when T cells were co-cultured with 10 nM peptide-loaded T2 tumor cells). (IFNγ: 104-fold, IL-2: 110-fold, TNFα: 81-fold, cell surface LAMP1: 40-fold, all compared to vector controls, Figures 1C-1D).

[0221] T cell dysfunction was observed in both CD8 and CD4 T cells. Furthermore, when comparing the degree of T cell activity inhibition in gene-modified cell populations with different levels of EGFP reporter expression, a correlation was found between Zap70-PD1 expression and inhibitory activity, suggesting that regulating Zap70-PD1 protein levels may be useful for regulating T cell function.

[0222] To achieve pharmacological control of Zap70-PD1 protein concentration, we subsequently generated fusion proteins with small molecule regulatory protein stability domains (e.g., self-cleaving degron (SED), where SED comprises an inhibitory protease, a homologous cleavage site and degron sequence, or a proteolytic target chimeric (Protac) binding domain). The combination of a phosphorylated ITAM-binding SH2 domain, an immunoreceptor tyrosine-based switch motif (ITSM), preferably an ITSM and an immunoreceptor tyrosine-based inhibitory motif (ITIM), and a small molecule regulatory protein stability domain is also referred to herein as CRASH-IT (Chemically Regulated and SH2-delivered Inhibitory Tail). Figure 2A-B shows a CRASH-IT embodiment that includes a small molecule-assisted shutoff tag (SMASh tag, Chung et al. Nat Chem Biol. 2015 Sep;11(9):713-20) as a SED. The SMASh tag consists of HCV NS3 / 4A protease and degron, which causes rapid proteolytic degradation by the proteasome. In the absence of an HCV protease inhibitor, the HCV protease cleaves the linker between the target protein and degron, thereby preventing protein degradation. On the other hand, in the presence of an HCV NS3 / 4A protease inhibitor, the entire fusion protein becomes a target of the proteasome and is degraded.

[0223] To verify whether the level of the Zap70-PD1 fusion protein could be controlled in this way, we created an N-terminal HA-tagged Zap70-PD1-SMASh fusion protein and introduced it into human T cells. Analysis of the fusion protein level by intracellular staining revealed that inhibition of protease activity with the HCV NS3 / 4A protease inhibitor asunaprevir reduced the fusion protein level in both primary human CD8 and CD4 cells, with maximum inhibition observed at around 0.2 μM, where the level was halved (Figure 2C-D).

[0224] Importantly, the Zap70-PD1-SMASh fusion protein retains the T cell activation inhibitory ability of the Zap70-PD1 fusion protein, and this inhibition of T cell function can be reversed by adding asunaprevir to both primary human CD8 T cells (Figure 2E) and CD4 T cells (Figure 2G).

[0225] Specifically, compared to DMSO-treated cells, activation of CD8+ T cells by peptide-loaded target cells (10 nM) was enhanced 8.8-fold, 11.5-fold, 6.3-fold, and 6.6-fold in IFNγ, IL2, TNFα, and cell surface LAMP1 expression, respectively. As a control, the effector function of Zap70-PD1-SMASh fusion protein-negative T cells (vector control) was not altered by the protease inhibitor (Figures 2F and 2H). We note that even with modest effects on fusion protein levels (Figure 2C), asunaprevir had a significant impact on the function of Zap70-PD1-SMASh-modified T cells (Figure 2E), indicating that the signal amplification characteristics of the TCR signaling pathway are highly sensitive to signal intensity.

[0226] The safety switch used in adoptive T-cell therapy should ideally be dose-adjustable and reversible. By analyzing the effect of increasing asunaprevir concentration in co-culture of antigen-loaded target cells and CDK4-specific T cells modified with the Zap70-PD1-SMASh switch, it was shown that T cell functionality is restored in a dose-dependent manner (Figure 3A-C), demonstrating that T cells can be regulated to reach the desired antigen sensitivity. Next, to determine whether the CRASH-IT platform acts as a reversible regulator of T cells, switching them from an active to an inactive state, asunaprevir-treated and control-treated T cells were washed and cultured for 72 hours in the absence of the drug. Subsequently, the T cells were either treated again with asunaprevir or left untreated and brought into contact with antigen-loaded target cells (experimental chart in Figure 3A). Notably, Zap70-PD1-SMASh-modified T cells showed substantial activity only when exposed to asunaprevir during the tumor co-culture period, regardless of whether the cells had been previously exposed to asunaprevir. In other words, activating the CRASH-IT switch beforehand does not change the outcome during subsequent use, demonstrating the platform's reversibility (Figure 3D).

[0227] The restoration of T cell function by small molecules was also demonstrated in co-culture experiments using Mel526 and NKIRTIL006 melanoma cells that endogenously express mutant CDK4 nascent antigen, but there was no effect on control cells MM90904 melanoma that express the wild-type CDK4 gene (data not shown).

[0228] To elucidate the characteristics of the protein domain essential for achieving reversible T cell inhibition, we compared SMASh-tagged Zap70 2xSH2 and Zap70-PD1 with SMASh-tagged Zap70-PD1 lacking the degron domain (data not shown). As observed with protein switches lacking the SMASh domain (Figure 1), the presence of the used PD1 tail is crucial for achieving strict control of TCR signaling, and the presence of degron, which induces proteasomal degradation, is necessary for the restoration of T cell activity triggered by the CRASH-IT switch.

[0229] Next, the SMASh tag is FKBP12 F36V We investigated the activation of the CRASH-IT platform via PROTAC by substituting the domain (Figure 4A-B). In the presence of heterologous functional dTAG-13 molecules, dimerization with the CRBN E3 ligase complex is induced, and FKBP12 F36V The fusion protein is rapidly degraded by the proteasomal (Nabet et al, Nat Chem Biol, 2018 May;14(5):431-441). Low-molecular-weight setting experiments showed that activation of the CRASH-IT platform using dTAG-13 PROTAC peaked at around 0.5 μM, while HCV NS3 / 4A inhibitors required significantly higher concentrations to achieve a similar activation level (Figure 4C-E). Furthermore, the SMASh tag was modified to FKBP12 F36V Domain substitution reduced basal T cell activation in tumor co-cultures when the small molecule was absent. FKBP12 F36V The reason for this may be that it is 591 bp shorter than the SMASh tag, resulting in a higher gene expression level. CDK4 TCR and Zap70-PD1-FKBP12 F36V (EGFP) cotransfected cells were selected based on EGFP and CD8 expression, and then developed in a culture medium containing dTAG-13 and high levels of IL-2 to create a T cell pool for cytotoxicity testing. The selected CD8 cells were then treated with dTAG-13. 51The Cr-loaded NKIRTIL006 tumor cell killing ability was increased, but the activity of control vector-modified T cells remained unchanged (Figure 4F-G).

[0230] Next, the flexibility of the CRASH-IT switch system was verified using T cells modified with second-generation CARs. When human T cells modified with anti-CD19-CD28-CD3ζ chain CARs (Figure 5A) (Brentjens et al, Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5426-35) were co-cultured with CD19-positive large (Raji) or Daudi (Figure 5B), cytokine production and degranulation of these cells were efficiently suppressed with Zap70-PD1-SMASh, and the function of these T cells was restored by the addition of asunaprevir (Figure 5C-F).

[0231] Similarly, the functional activity of CD8 T cells modified with NY-ESO-1 co-antigen-specific TCRs (Linnemann et al, Nat Med. 2013 Nov;19(11):1534-41) was inhibited by the CRASH-IT switch and restored by the addition of asunaprevir (Figure 6).

[0232] Finally, when the endogenous TCR complex of primary human T cells was stimulated with plates coated with anti-CD3 antibody or anti-CD3 / CD28 antibody, T cell function was again suppressed by CRASH-IT and restored by drug addition (data not shown).

[0233] Proteolysis by asnaprevir was more efficiently performed in CD8 cells than in CD4 cells. For this reason, the restoration of T cell function by asnaprevir was more completely achieved in CD8+ T cells than in CD4+ T cells. To explore the possibility of creating a displacement CRASH-IT switch system with a dynamic range optimized for different immune cell types, a modified version of Zap70-PD1-SMASh (Figure 7A), which shows slightly less stringent T cell suppression in the absence of asnaprevir in CD8 cells, was created. The adjusted tuZap70-PD1-SMASh switch retained the ability to efficiently suppress the function of CD4 T cells modified with the CDK4 TCR, but the restoration of T cell function by the addition of asnaprevir was significantly improved (Figure 7B). To test whether this switch system could also be used to control CD4+ T cell recognition via HLA class II, primary human CD4+ T cells were co-transduced with the tuZap70-PD1-SMASh switch and an HLA class II-restricted CMV TCR, and the resulting cells were co-cultured with peptide-loaded CBH 5477 target cells. The introduction of the tuZap70-PD1-SMASh switch reduced antigen sensitivity by approximately one-thousandth, and the function of CD4+ T cells was almost completely restored by asnaprevir treatment (Figures 7C-D). This demonstrates that a CRASH-IT system optimized for a specific cell type can be constructed. tuZap70-PD1-SMASh was obtained by adding an alanine amino acid residue encoding DNA to the N-terminus after the start codon (the valine amino acid residue also showed similar results in the test).

[0234] Conceptually, CRASH-IT includes functional elements that induce access to antigen receptors, provide inhibitory signals, and offer the possibility of regulating the strength of these inhibitory signals. In the design we developed, these functional elements are formed by the Zap70 SH2 domain, the PD1 tail, and a small molecule-controlled protein stability domain (SMASh tag or FKBP12 F36V ) respectively.

[0235] Furthermore, by including different inhibitory domains from receptors containing ITIM and ITSM, it is possible to alter the level of T cell suppression and direct that suppression to specific T cell output signals. Figures 8 and 9 demonstrate that constructs containing different ITSM and ITIM motifs from other inhibitory tails can indeed improve the stringency of the chimeric polypeptide and system according to the present invention. All tested domains contain both ITSM and ITIM, except for LY9, which consists of only two ITSM domains and does not contain an ITIM domain (see also Figure 11). In addition, Figure 10 shows that alternative SH2-containing docking domains can be used in the present invention. All of the surrogate SH2 domains tested interact with phosphorylated ITAM (see also Figure 12) within the context of the present invention (Katsuyama et al, Front Immunol. 2018; 9: 1088, Ngoenkam et al, Immunology. 2018 Jan; 153(1): 42-50, Koch et al, Trends Immunol. 2013 Apr;34(4):182-91).

[0236] Furthermore, the broad applicability of the CRASH-IT platform was demonstrated in NK cells as well. The NK cell line KHYG-1 can effectively recognize and kill HLA class I and class II deficient K562 tumors using its endogenous NK cell activating receptor (Suck et al, Exp Hematol. 2005 Oct;33(10):1160-71). Zap70-PD1-FKBP12 F36V When the switch was expressed, it efficiently suppressed IFNγ, IL2, and TNFα production in NK cells co-cultured with K562 tumors in the absence of drugs, and this suppression was released in the presence of dTAG-13 PROTAC (Figure 13).

[0237] Current CAR designs often include ITAMs containing the CD3 ζ chain signaling domain, but alternative ITAM-containing signaling domains have also been reported, such as fragments of the CD3 ε chain (Nolan et al, Clin Cancer Res, 1999 Dec;5(12):3928-41), the immunoglobulin receptor γ (gamma) chain FcεRI (Ren-Heidenreich et al, Cancer Immunol Immunother. 2002 Oct;51(8):417-23), and DAP12 (Topfer et al, J Immunol. 2015 Apr 1;194(7):3201-12). We tested the compatibility of the CRASH-IT switch with a panel of CARs containing alternative ITAM-containing signaling domains and demonstrated inhibition of T cell function in the absence of drugs and restoration of T cell function upon drug addition in T cells co-expressing different CARs with the CRASH-IT switch (Figure 14).

[0238] Embodiments of CRASH-IT utilizing the SMASh domain include HCV-derived protein sequences that may potentially be immunogenic within the immune host. FKBP12 F36V Further embodiments of the domain-based CRASH-IT platform are controlled by PROTAC molecules such as dTAG-13. However, the size of dTAG-13 (molecular weight: 1049.18) may limit its oral availability.

[0239] Therefore, we evaluated whether it is possible to combine the CRASH-IT switch system with another protein stability control domain controllable by small molecule compounds that have demonstrated oral bioavailability and are used clinically. We found that a CRASH-IT embodiment comprising a CRBN polypeptide substrate domain that binds to the CRBN protein in response to a drug and thereby promotes ubiquitin pathway-mediated degradation of the chimeric polypeptide can be successfully incorporated into the present invention. This is illustrated using a CRASH-IT embodiment comprising a first portion comprising an SH2 domain from a protein that binds to an activation motif based on phosphorylated immunoreceptor tyrosine, preferably a switch motif based on immunoreceptor tyrosine (ITSM), preferably a third portion comprising an inhibitory motif (ITIM) based on both ITSM and immunoreceptor tyrosine, and a second portion comprising a zinc finger-based degron (i.e., a portion comprising a small molecule controllable protein stability domain). Such embodiments of CRASH-IT have been successfully controlled by immunomodulatory imide drugs (IMiDs, also known as cerebron modulators), including clinically approved and orally administered molecules such as thalidomide, lenalidomide, and pomalidomide.

[0240] Thalidomide (molecular weight: 258.23), lenalidomide (molecular weight: 259.26), and pomalidomide (molecular weight: 273.24) are immunomodulatory drugs (IMiDs) clinically used to treat multiple myeloma. Previous studies have shown that the activity of these compounds depends, for example, on the CRBN E3 ligase-dependent degradation of the IKZF1 protein. Furthermore, it has been found that a 23-amino acid zinc finger motif (IKZF1 zinc finger 2, ZF2) within the IKZF1 sequence constitutes the minimal degron (or CRBN polypeptide substrate domain) for thalidomide, lenalidomide, and pomalidomide-dependent proteolysis. Combining the IKZF1 ZF2 sequence with an adjacent zinc finger 3 (IKZF1 ZF3) that interacts with the CRBN-IKZF1 ZF2 interface yields an improved IKZF1-derived degron approximately 57 amino acids long (Sievers et al, Science. 2018 Nov 2;362(6414). pii: eaat0572). This improved degron sequence is fused to the target protein, for example, so that the stability of the resulting protein can be controlled by the addition of thalidomide, lenalidomide, pomalidomide, or any other IMiD, similar to other CRBN polypeptide substrate domains that can bind to CRBN proteins in response to drugs (Koduri et al, Proc Natl Acad Sci US A.2019 Feb 12;116(7):2539-2544).

[0241] Pomalidomide and lenalidomide are second-generation IMiDs and differ from thalidomide in that they have an aniline group on the solvent-exposed phthalimide ring at the C4 position. The presence of this aniline group is thought to improve the efficacy of lenalidomide and pomalidomide compared to thalidomide. While the therapeutic effects and side effects (myelosuppression, anti-inflammatory effects, T-cell costimulation, NK cell proliferation, angiogenesis, and teratogenicity) of these three drugs overlap, the degree of these effects differs among them. The recommended starting doses for thalidomide, lenalidomide, and pomalidomide are 200 mg / day, 25 mg / day, and 4 mg / day, respectively, reflecting the differences in their potency.

[0242] To control T cell activity levels with IMiD, we created a CRASH-IT switch containing the Zap70 2xSH2 domain, PD1 signaling domain, and optimal amounts of degron in IKZF1 ZF2-ZF3 (Figures 15-16). Primary T cells were co-introduced using a CDK4 nascent antigen-specific TCR and an IKZF1-based CRASH-IT switch, and co-cultured with NKIRTIL006 tumor cells endogenously expressing the CDK4 nascent antigen. In the absence of IMiD, cytokine production and LAMP-1 expression on the cell surface were reliably suppressed, and activity levels were reliably restored in the presence of pomalidomide or lenalidomide, but the degree of recovery was low in the presence of thalidomide (Figure 16).

[0243] The anti-inflammatory effects of IMiDs can inhibit the activity of cell therapies if high drug concentrations are required to inactivate the safety switch. Therefore, there is a need to create artificial deglons that show high affinity for IMiDs and can control the safety switch at drug concentrations that reduce the degradation of endogenous targets. It has been previously reported that a complex zinc finger motif containing the β-turn sequence of ZFP91 ZF4 and the α-helix sequence of IKZF1 ZF2 shows higher affinity for thalidomide compared to the parent sequences ZFP91 ZF4 and IKZF1 ZF2 (Sievers et al, Science. 2018 Nov 2;362(6414). pii: eaat0572). It has also been separately reported that the IKZF1 ZF2-3 sequence shows superior protein resolution compared to the IKZF1 ZF2 sequence, but the IKZF1 ZF3 sequence is not included in the zinc finger deglon in this analysis.

[0244] To generate degrons that interact with thalidomide with high affinity, the β-turn sequence of IKZF1 ZF2 was replaced in IKZF1 ZF2-3 degrons with β-turn sequences from various zinc fingers (ZNF653 ZF4, ZFP91 ZF4, ZNF276 ZF4, ZNF827 ZF1). Analysis of T cell activity as a function of drug concentration revealed that complex zinc finger grafts containing ZNF653, ZFP91, ZNF276, and ZNF827 β-turn grafts could be inactivated by reducing the concentration of lenalidomide, pomalidomide, or thalidomide, and drug sensitivity was significantly improved, particularly with the latter (Figure 16). Specifically, the thalidomide concentration required to achieve the same level of cytokine production (IFNγ, IL-2, or TNFα) or surface LAMP-1 expression was reduced to approximately 1 / 1000th in T cells expressing chimeric degrons compared to those expressing parental IKZF1 ZF2-3 degrons (Figure 16D). Therefore, Zn finger-based CRASH-IT switches such as Zap70-PD1-ZFP91 / IKZF1 can be used to control T cell function at thalidomide concentrations that minimize thalidomide binding to their normal ligands.

[0245] Of note is that in these IMiD dose setting experiments, effective T cell reactivation was observed even at high concentrations of the drug. On the other hand, FKBP12 F36V Using high concentrations of drugs via a switch based on / dTAG-13 results in suboptimal T cell function reactivation. Suboptimal degradation of protein targets at high PROTAC concentrations has been previously reported, involving E3 ligase-PROTAC and PROTAC-target protein (FKBP12). F36V This is thought to be due to the so-called "hook effect" (An et al, EBioMedicine. 2018 Oct;36:553-562), in which the dimers of (e.g.) control the intended E3 ligase-PROTAC-target protein trimer. Since there is no significant hook effect with the combination of zinc fingers and IMiD, optimal T cell reactivation can be achieved in both Cmax and Cmin, making the clinical use of IMiD easier. Finally, for example, FKBP12F36V Compared to a domain (107 amino acids), the size of a zinc finger-based degron (approximately 57 amino acids) is smaller, enabling compact vector designs, including the design of a single vector system in which CAR / TCR and CRASH-IT switch expression are linked.

[0246] Finally, we compared the efficiency of Zap70-PD1-zinc finger constructs containing or not containing IKZF1 ZF3 (dual ZF and single ZF, respectively), or wild-type dual zinc finger constructs derived from IKZF1, IKZF3, ZFP91, ZNF276, and ZNF653 (Figure 17A). The results showed that the activity of T cells expressing CDK4 TCR and Zap70-PD1-ZFP91 / IKZF1-zinc finger dual ZF degrons could be restored most efficiently in the presence of IMiDs (Figures 17B-E).

[0247] The chimeric polypeptide and system according to the present invention, CRASH-IT, is a volume-configurable and reversible T cell and NK cell safe switch platform that is independent of the properties of the activating antigen receptor, as demonstrated by its use with high-affinity and intermediate-affinity class I restriction TCRs, class II restriction TCRs and CARs in the context of CD4 T cells, CD8 T cells, and NK cells.

[0248] CRASH-IT's flexibility is particularly valuable in environments where precise control of T cell and NK cell sensitivity is desired. Furthermore, the combination of CRASH-IT with existing CARs does not require structural redesign of the CAR, making it especially valuable for CAR designs that have already been evaluated in clinical trials. Materials and methods Retroviral DNA constructs

[0249] All DNA constructs were generated using the MP71 retroviral expression vector backbone (Engels et al, Hum Gene Ther. 2003 Aug 10;14(12):1155-68). Briefly, codon-optimized DNA sequences were synthesized as gene fragments by IDT and cloned into the MP71 vector by Gibson assembly (Gibson et al, Nat Methods. 2009 May;6(5):343-5). To generate the MP71-Zap70 2xSH2-PD1 tail IRES-EGFP vector, codon-optimized sequences encoding human Zap70 fragments (P43403, 1-264 aa), GGS linker, human PD1 intracellular domain (Q15116, 192-288 aa), stop codons, and an IRES-EGFP reporter were cloned into MP71.

[0250] Other constructs containing the MP71 IRES-EGFP scaffold were constructed from the following coding sequences: MP71-Zap70-2xSH2 domain-IRES-EGFP: human Zap70 (P43403, 1-264 aa), MP71 PD1-tail-IRES-EGFP: MV coding sequence (i.e., the start codon methionine and additional valine that generates the Kozak sequence), and human PD1 intracellular domain (Q15116, 192-288 aa). MP71-Zap70-PD1-SMASh-IRES-EGFP: Human Zap70 (P43403, 1-264aa), GGS linker, human PD1 intracellular domain (Q15116, 192-288aa), SGGGS linker, and SMASh tag at 304aa (Chung et al, Nat Chem Biol. 2015 Sep;11(9):713-20). MP71-Zap70 2xSH2-SMASh-IRES-EGFP: Human Zap70 (P43403, 1-264 aa), SGGGS short linker, and SMASh tag at 304 aa. MP71-IRES-EGFP vector control: Unrelated 248 aa Tet-On 3G transactivator (Clontech). MP71-HA-Zap70-PD1-SMASh-tag-IRES-EGFP: The HA tag (MVYPYDVPDYAGSGV) coding sequence is followed by the Zap70-PD1-SMASh coding sequence. MP71-tuZap70-PD1-SMASh-IRES-EGFP: DNA encoding an alanine residue is added after the start codon of MP71-Zap70-PD1-SMASh-IRES-EGFP. MP71-Zap70-PD1-SMASh-δ-IRES-EGFP (Degron sequence deletion): The DNA encoding the last 78aa of the SMASh tag in the MP71-Zap70-PD1-SMASh-IRES-EGFP construct is deleted.MP71 CD19 ScFv-CD28-CD3 ζ CAR-IRES-huEGFRt: A second-generation CD19-specific CAR was subcloned from the SFG-19-28z vector (Brentjens et al. Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5426-35) to the MP71 skeleton by Gibson assembly, together with an IRES-cleaved human EGFR (huEGFRt) reporter (Wang et al. 2011). MP71-IRES-huEGFRt vector control: The CAR insert in the MP71 CD19 ScFv-CD28-CD3 ζ CAR-IRES-huEGFRt vector was replaced with an unrelated 248 aa Tet-On 3G trans-activated protein coding sequence (Clontech). The PD1 cytoplasmic domain (PD1 tail) coding sequence in the MP71-Zap70-PD1-SMASh-IRES-EGFP vector is replaced with the DNA sequence encoding the cytoplasmic domain using BTLA (Q7Z6A9, 179-289 aa), SIRPA (P78324, 395-504 aa), Siglec 5 (O15389, 463-551 aa), Siglec 9 (Q9Y336, 370-463 aa), Siglec 11 (Q96RL6, 585-698 aa), PECAM1 (P16284-1, 621-738 aa), and LY9 (Q9HBG7, 477-655 aa) to construct a CRASH-IT embodiment encoding the corresponding inhibitory cytoplasmic domain. The Zap70 2xSH2 domain encoding sequence (P43403, 1-264aa) in the MP71-Zap70-PD1-SMASh-IRES-EGFP vector was replaced with a DNA sequence encoding either Syk 2xSH2 (P43405-1, 1-287aa) or Lck SH4-Unique-SH3-SH2 (P06239-1, 1-254 aa) to construct a CRASH-IT embodiment encoding the corresponding SH2 domain. The SMASh domain encoding sequence in the MP71-Zap70-PD1-SMASh-IRES-EGFP vector was replaced with FKBP. F36VWe constructed a CRASH-IT embodiment controllable by dTAG-13 PROTAC by substituting with the DNA sequence encoding (Nabet et al. Nat Chem Biol. 2018 May;14(5):431-441).

[0251] To generate a CAR-T panel encoding alternative ITAM-containing domains, the CD3 ζ chain of the MP71 CD19 ScFv-CD28-CD3 ζ CAR-IRES-huEGFRt vector was replaced with an ITAM containing a cytoplasmic domain of either the γ chain of the immunoglobulin receptor FcεRI (FCER1G) (P30273, 45-86 aa), the CD3 ε chain (P07766, 153-207 aa), or DAP12 (O43914, 62-113 aa). Alternatively, the CD3 ζ chain was deleted to generate a CAR construct without an ITAM-containing domain (negative control). The MP71 CD19 ScFv-CD28-CD3 ζ CAR-IRES-huEGFRt vector was prepared by substituting the CD28-CD3 ζ coding sequence with the full-length CD3 ε-chain sequence CD3E (P07766, 25-207 aa).

[0252] Constructs containing various zinc finger deglons were created in MP71 Zap70-PD1-Zinc finger IRES-EGFP format using IKZF1 ZF2-3 deglon-containing sequences (Q13422, 141-197 aa), IKZF3 ZF2-3 deglon-containing sequences (Q9UKT9, 142-198 aa), ZFP91 ZF4-5 deglon-containing sequences (Q96JP5, 396-455 aa), ZNF276 ZF4-5 deglon-containing sequences (Q8N554, 520-579 aa), ZNF653 ZF4-5 deglon-containing sequences (Q96CK0, 552-611 aa), or ZNF692 ZF4-5 deglon-containing sequences (Q9BU19, 413-473 aa). For CRASH-IT switches encoding composite zinc finger deglons, the IKZF1 ZF2 β-turn sequence (FQCNQCGASFT) was replaced with the β-turn sequences of ZNF653 ZF4 (LQCEICGYQCR), ZFP91 ZF4 (LQCEICGFTCR), ZNF276 ZF4 (LQCEVCGFQCR), and ZNF827 ZF1 (FQCPICGLVIK). For CRASH-IT switches encoding ZFP91 / IKZF1 composite zinc finger (single ZF) without IKZF1 ZF3, the IKZF1 ZF3-containing sequence (Q13422, 170-197 aa) was deleted from the Zap70-PD1-ZFP91 / IKZF1 composite zinc finger (double ZF) construct.

[0253] The HLA class I-restricted CDK4 TCR (TCR 17, Stronen et al, Science. 2016 Jun 10;352(6291):1337-41) and NY-ESO-1 TCR (TCR 1, Linnemann et al, Nat Med. 2013 Nov;19(11):1534-41) have been described previously. The variable domain sequences of the HLA class II-restricted CMV-pp65 TCR (van Loenen et al, PLoS One. 2013 May 30;8(5):e65212) were kindly provided by M.H. Heemskerk (LUMC, NL) and cloned into the TCR flex MP71 vector (Linnemann et al., Nat Med. 2013 Nov;19(11):1534-41). Cell lines and cell culture

[0254] FLYRD18, T2, MM90904 (provided by Marco Donia, Herlev Hospital, Denmark), Mel526 (Stronen et al, Science. 2016 Jun 10;352(6291):1337-41), NKIRTIL006 (Kvistborg et al, Oncoimmunology. 2012 Jul 1; 1(4): 409-418), K562, Daudi, Raji and CBH 5477 (provided by M.H. Heemskerk) cells were cultured in IMDM (Invitrogen, #21980065) supplemented with 8% FCS (Invitrogen, #F7524-500ML) and penicillin-streptomycin (100 IU / ml penicillin, 100 μg / ml streptomycin, Sigma-Aldrich, #11074440001). FLYRD18, MM909, Mel526 and NKIRTIL006 cells were passaged every 2-3 days using trypsin-EDTA (Invitrogen, #15400054).

[0255] The human NK cell line KHYG-1 (DSMZ, Leibniz, Germany) was cultured in RPMI supplemented with 8% FBS and penicillin-streptomycin (100 IU / ml penicillin, 100 μg / ml streptomycin) containing 500 IU / ml IL-2 (Novartis). All cell lines were tested for mycoplasma by PCR-based screening (Young et al, Nat Protoc. 2010 May;5(5):929-34) and confirmed to be negative. Production of retrovirus

[0256] Retroviral particles were produced in FLYRD18 packaging cells. Briefly, one day before transfection, 700,000 FLYRD18 packaging cells per 10 cm culture dish were seeded. The next day, the cell culture medium was refreshed with IMDM supplemented with 8% FCS without antibiotics. 25 μl of X-tremeGENE 9 (Roche, #6365809001) was mixed with 800 μl of Opti-MEM (Invitrogen, #11058-021) and incubated at room temperature for 5 minutes. Then, the Optimem-X-tremeGENE 9 mixture was added to 10 μg of retroviral plasmid DNA dissolved in water, incubated at room temperature for 15 minutes, and the resulting transfection mixture was added dropwise to the packaging cells. The supernatant containing retrovirus was collected 48 hours after transfection and used immediately or snap-frozen in liquid nitrogen. Isolation and activation of T cells

[0257] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats of healthy donors (Sanquin (Amsterdam, NL)) by Ficoll-Isopaque density centrifugation (Linnemann et al, Nat Med. 2013 Nov;19(11):1534-41) and cryopreserved until use. To generate an activated T cell population, PBMCs were thawed in PBS solution containing 5% FCS, counted, and mixed with CD3 / CD28 Dynabeads (CTS, #40203D) at a 1:1 cell-to-bead ratio and adjusted to a density of 7 10 cells / ml.

[0258] After tumble-instilling at room temperature for 30 minutes, the mixture was placed on a magnet to remove unbound cells. The bead-bound T cells were resuspended in penicillin-streptomycin-supplemented RPMI containing 10% human serum (Sigma-Aldrich, #H3667-100ML) and 100 IU / ml IL-2 (Novartis) and 5 ng / ml IL-15 (Peprotech, #200-15), and 0.75 × 10⁶ 6 Seeds were seeded at a density of cells / ml. Rotational plasma transfer of T cells and NK cells

[0259] A 24-well untreated cell culture plate was coated overnight at 4°C with 10 μg / ml retronectin (Takara, #T100B). The following day, the retronectin solution was removed, and the wells were blocked for 30 minutes with PBS containing 2% BSA (Sigma-Aldrich, A9418-500g). Activated T cells (RPMI / 10% human serum / penicillin-streptomycin / 200 IU / ml IL-2 and 10 ng / ml IL-15) were treated with 0.25 × 10⁶ cells. 6 Cells / ml) or KHYG-1 NK cells (RPMI / 8% FCS / penicillin-streptomycin / 1000 IU / ml IL-2 0.25×10 6 Cells ( / ml) were mixed with retroviral supernatant in a 1:1 ratio (volume / volume) in a retronectin-coated 24-well plate and centrifuged at 2000 RPM for 90 minutes at room temperature. Peptide support

[0260] To use as a T cell target, T2 and CBH 5477 cells were loaded with IMDM solutions of the indicated concentrations of HLA-A*02:01 restriction mutant CDK4 peptide (ALDPHSGHFV), HLA-A*02:01 restriction NY-ESO-1 peptide (SLLMWITQA), or HLA-DR1 restriction CMV peptide (KYQEFFWDANDIYRI) at 37°C for 1 hour. The cells were then washed once and used in co-culture experiments. Cytokine release assay and antibody staining

[0261] T cells and NK cells were cultured using the indicated concentrations of asunaprevir (MedChemExpress, #HY-14434), grazoprevir (MedChemExpress, #HY-15298, dTAG-13 (Tocris, #6605)), or DMSO control in T cell culture medium (RPMI / 10% human serum / penicillin-streptomycin, 100 IU / ml IL2 and 5 ng / ml IL15) or NK cell culture medium (RPMI / 8% FCS / penicillin-streptomycin, 500 IU / ml) Each cell was pre-treated for 24 hours before the co-culture experiment using IL2). 100,000 T cells or NK cells and 100,000 indicator tumor cells were mixed in a 96-well plate with T cell or NK cell culture medium containing Golgi plugs (1:1000 dilution, BD, #51-2301KZ) and anti-LAMP1-APC (1:100 dilution, Biolegend, #328620) in the presence of the indicator drug or DMSO control, and left at 37°C for 5 hours.

[0262] After standing at constant temperature, the cells were washed once with PBS and stained with a 1:400 diluted IR dye (Invitrogen, #L34976) at 4°C for 5 minutes. Subsequently, the cells were washed once with FACS buffer (PBS + 0.5% BSA) and stained with anti-CD8-PerCP Cy5.5 (1:20 dilution, BD, #341050), anti-CD4 BV711 (1:50 dilution, Biolegend, #317440), anti-Moor constant TCR-PE (1:200 dilution, BD, #553172 in experiments using introduced TCRs), or cetuximab-PE (1:200 dilution, R&D Systems, #FAB9577P in experiments using introduced CAR constructs containing a truncated human EGFR reporter) at 4°C for 20 minutes. Subsequently, the cells were washed once with FACS buffer and fixed at 4°C for 20 minutes using BD fixation / permeabilization solution (#51-2090KZ).

[0263] After permeabilization, the cells were washed twice with BD permeabilization and washing buffer (#51-2091KZ) and stained at 4°C for 20 minutes with anti-IFNγ-BV421 (1:100 dilution, BD, #564791), anti-IL-2-PE-Cy7 (1:100 dilution, BD, #560707), and anti-TNFα-BV650 (1:100 dilution, Biolegend, #502938), all diluted with permeabilization and washing buffer. The cells were then washed twice, resuspended in 100 μl of FACS buffer, and directly analyzed using a custom Fortessa analyzer. Data were analyzed using FlowJo and Prism 7 software.

[0264] Similarly, T cells were intracellularly stained with anti-HA-AF647 (1:200 dilution, Cell Signaling Technology, #3444S), and K562, Raji, and Daudi tumor cells were surface-stained using anti-CD19-PE (1:200 dilution, BD, #345789) or an isotype control (Biolegend, #400111) as described above. T cell selection and rapid proliferation

[0265] Zap70-PD1-FKBP F36V Add the culture medium of the expressing cells from one day before cell sorting. 51 Supplement with 0.5 μM dTAG-13 PROTAC up to 4 days before the Cr test. CDK4 TCR and Zap70-PD1-FKBP F36V Primary human T cells modified with either a switch or an IRES-EGFP vector control were sorted using a Beckman-Coulter Moflo Astrios with an 80 μM nozzle. The cells were cultured for 7 days under standard T cell culture conditions of RPMI / 10% human serum / penicillin-streptomycin 100 IU / ml IL-2 and 5 ng / ml IL-15.

[0266] After this, the cells were grown using the rapid expansion protocol (REP). In short, 2 x 10⁶ cells were obtained from three donors. 8A mixture of individual feeder cells was irradiated at 4,000 rad to produce (Gammacell 40 Exactor), and the resulting feeder cells were 1 × 10⁶ 6 Selected T cells were mixed with IL-2 (final concentration 3000 IU / ml) in 150 ml of 20 / 80 T cell mixed culture medium (Invitrogen, #041-96658P) supplemented with 4.5 μg of OKT3 (Invitrogen, #16-0037-85) and penicillin-streptomycin (100 IU / ml penicillin, 100 μg / ml streptomycin). On day 6, the culture medium was refreshed with 3000 IU / ml IL-2-containing culture medium, and the cells were cultured in two separate cultures every three days in IL-2 (final concentration 3000 IU / ml)-containing culture medium. On day 12, 51 For three days before use in Cr testing, the cells were switched to standard T-cell culture conditions (RPMI / 10% human serum / penicillin-streptomycin / 100 IU / ml IL-2 and 5 ng / ml IL-15). 51 Cr test

[0267] 51 One day prior to Cr testing, T cells were pre-treated with 0.5 μM dTAG-13 PROTAC or DMSO under standard T cell culture conditions of RPMI / 10% human serum / penicillin-streptomycin / 100 IU / ml IL-2 and 5 ng / ml IL-15. 5 × 10 5 Resuspend individual tumor cells in 100 μl of culture medium, and add 100 μCi 51 The cells were gently mixed with Cr and left to stand at 37°C for 45 minutes. In parallel, 100 μl of diluted T cell solution treated with either dTAG-13 or DMSO control was dispensed into 96-well plates. 100 μl of culture medium alone (spontaneous release) and 100 μl of 1% Triton solution (maximum release) were used as controls.

[0268] After labeling, the target cells were washed three times with 1 ml of culture medium. The labeled target cells were resuspended at 50,000 cells / ml and added to 96-well plates at a rate of 100 μl per well. The plates were then centrifuged at 900 rpm for 2 minutes and left to stand at 37°C for 4 hours. After culturing, 50 μl of the supernatant was added to Lumaplate-96 (Packard Bioscience, #6005164), dried overnight, and counted using PerkinElmer, TopCount NXT. Experimental values ​​were normalized using spontaneous release and maximum release controls.

[0269] Having fully described the present invention, it goes without saying that those skilled in the art can carry out the same thing within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention or without excessive experimentation.

[0270] All documents cited herein (including journal articles or abstracts, published or corresponding patent applications, patents, or other documents) are fully incorporated herein, including all data, tables, figures, and text presented in those documents. Furthermore, the entire content of documents cited within documents cited herein is also fully incorporated by reference.

[0271] Any description of known methods, prior art, or known or prior art does not constitute in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

[0272] The above description of specific embodiments will make the general nature of the present invention sufficiently clear, so that others, by applying the knowledge of those skilled in the art (including the contents of the references cited herein), can readily modify and / or adapt such specific embodiments to various uses without excessive experimentation and without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and equivalence of the disclosed embodiments, based on the teachings and advice presented herein, etc.

[0273] Naturally, the terms and phrases in this specification are for illustrative purposes only, and not to be limiting, to be interpreted by a person skilled in the art in conjunction with the teachings and advice provided herein and their knowledge.

Claims

1. A cell comprising a chimeric polypeptide or a nucleic acid comprising a polynucleotide encoding said chimeric polypeptide, said chimeric polypeptide comprising a) a first portion comprising an SH2 domain derived from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); b) a second portion comprising a small molecule regulatory destabilization domain, The small molecule regulatory destabilization domain comprises: i) a self-cleaving degron (SED) containing an inhibitory protease, a cognate cleavage site, and a degron sequence; ii) a proteolysis targeting chimera (Protac) binding domain, and iii) a CRBN polypeptide substrate domain that binds to a CRBN protein in response to a drug, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide; the second moiety selected from the group consisting of: c) a third portion comprising an immunoreceptor tyrosine-based switch motif (ITSM), or an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITSM; including, cells.

2. The cell of claim 1 , wherein the third portion comprises an ITSM and an ITIM.

3. The cell of claim 1 or 2, wherein the Protac comprises an E3 ubiquitin ligase binding group (E3LB), a linker, and a protein binding group that binds to the Protac binding domain in the chimeric polypeptide.

4. The cell of any one of claims 1 to 3, wherein the ITAM is an ITAM contained in a T cell receptor (TCR) complex, an NK cell receptor (NKR) complex, and / or a chimeric antigen receptor (CAR), and / or the cell further comprises a T cell receptor, a chimeric antigen receptor (CAR), and / or an NK cell receptor (NKR).

5. The cell according to claim 4 , wherein the ITAM is derived from the CD3 ζ chain, the CD3 ε chain, the CD3 δ chain, the CD3 γ chain, the γ chain of the immunoglobulin receptor FcεRI, and DAP12.

6. The cell of claim 4 or 5, wherein the cell is a T cell, a CAR T cell, an NK cell, and / or a CAR NK cell.

7. The cell of any one of claims 1 to 6, wherein the SH2 domain is derived from a protein selected from the group consisting of Zap70, Syk, and Lck, and / or the chimeric polypeptide comprises one or more SH2 domains derived from a protein that binds to a phosphorylated immunoreceptor tyrosine-based activation motif.

8. The cell of any one of claims 1 to 7, wherein the ITIM and / or ITSM is derived from an inhibitory receptor protein.

9. The cell of any one of claims 1 to 8, wherein the ITIM and / or ITSM is derived from an inhibitory immunoreceptor protein.

10. The cell of any one of claims 1 to 9, wherein the ITIM and / or ITSM is derived from a protein selected from the group consisting of PD1, BTLA, SIRPα, Siglec-5, Siglec-9, Siglec-11, PECAM1, or LY9.

11. the CRBN polypeptide substrate domain is a C2H2 zinc finger protein or a fragment thereof capable of drug-induced binding to the CRBN protein; and / or the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, ZFN654, ZNF787, ZNF653, ZFP91, ZNF276, ZNF827, or fragments thereof capable of drug-inducible binding to the CRBN protein; and / or 11. The cell of any one of claims 1 to 10, wherein the CRBN polypeptide substrate domain comprises a composite fusion polypeptide comprising at least a first fragment of a first C2H2 zinc finger protein and a second fragment of a second C2H2 zinc finger protein, and wherein the combination of the first fragment and the second fragment in the composite fusion polypeptide is capable of drug-inducible binding to the CRBN protein.

12. The cell of claim 11, wherein the fragments of IKZF1, IKZF3, ZFN654, ZNF787, ZNF653, ZFP91, ZNF276, and ZNF827 capable of drug-inducible binding to the CRBN polypeptide are selected from the group consisting of IKZF1 ZF2-3 (SEQ ID NO: 41), IKZF3 ZF2-3 (SEQ ID NO: 42), ZFP91 ZF4-5 (SEQ ID NO: 43), ZNF276 ZF4-5 (SEQ ID NO: 44), ZNF653 ZF4-5 (SEQ ID NO: 45), and ZNF692 ZF4-5 (SEQ ID NO: 46).

13. The cell of claim 11 or 12, wherein the composite fusion polypeptide contained within the CRBN polypeptide substrate domain comprises a first fragment selected from the β-turns of ZFP91 ZF4 (LQCEICGFTCR; SEQ ID NO: 52), ZFN653 ZF4 (LQCEICGYQCR; SEQ ID NO: 53), ZNF276 ZF4 (LQCEVCGFQCR; SEQ ID NO: 54), and ZNF827 ZF1 (FQCPICGLVIK; SEQ ID NO: 55), and a second fragment selected from the α-helix of IKZF1 ZF2 (QKGNLLRHIKLH; SEQ ID NO: 56).

14. The cell of claim 13, wherein the composite fusion polypeptide comprises a β-turn of the ZFP91 ZF4 and an α-helix of the IKZF1 ZF2.

15. The cell according to any one of claims 11 to 14, wherein the composite fusion polypeptide comprises one selected from the amino acid sequences according to SEQ ID NOs: 47 to 51.

16. the CRBN polypeptide substrate binding domain comprises or further comprises IKZF1 ZF3 (FKCHLCNYACRRRDALTGHLRTH: SEQ ID NO: 57), and / or The cell of any one of claims 1 to 15, wherein the drug binds the CRBN polypeptide substrate domain to the CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide, and the drug is an IMiD.

17. The cell of claim 16, wherein the CRBN polypeptide substrate binding domain comprises a beta turn of the ZFP91 ZF4, an alpha helix of the IKZF1 ZF2, and an IKZF1 ZF3.

18. 17. The cell of claim 16, wherein the IMiD is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide), and CC-885.

19. a) the SH2 domain comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence according to SEQ ID NOs: 7 to 11, or comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence according to SEQ ID NOs: 12 to 14, b) the first part of the chimeric polypeptide comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence according to SEQ ID NOs: 7 to 11, or comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence according to SEQ ID NOs: 12 to 14, c) the ITAM is YxxL / Ix(6-8)YxxL / I, where x is any amino acid; d) the SH2 domain is an SH2 domain that binds to a phosphorylated ITAM contained in an amino acid sequence according to SEQ ID NOs: 1 to 6, or an SH2 domain that binds to an amino acid sequence having 90% or more identity to an amino acid sequence according to SEQ ID NOs: 1 to 6; e) the ITIM is S / I / V / LxYxxI / V / L, where x is any amino acid, and / or the ITSM is TxYxxV / I, where x is any amino acid; f) the ITSMs and / or ITIMs are ITSMs and / or ITIMs contained within the amino acid sequences according to SEQ ID NOs: 15 to 22; g) the third part of the chimeric polypeptide comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence according to SEQ ID NOs: 15 to 22; h) the second part of the chimeric polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 35 or 40 or SEQ ID NO: 41 to 57; and / or i) the chimeric polypeptide comprises an amino acid sequence having 90% or more identity to an amino acid sequence according to SEQ ID NO: 23-34, SEQ ID NO: 36, or SEQ ID NO: 58-68; A cell according to any one of claims 1 to 18.

20. 20. The cell of any one of claims 1 to 19, wherein the first portion is located at the N-terminus of the chimeric polypeptide, the second portion is located at the C-terminus of the chimeric polypeptide, and the third portion is located between the first portion and the second portion.

21. the order of the first part (P1), the second part (P2), and the third part (P3) is xP1xP3xP2; 20. The cell of any one of claims 1 to 19, wherein x at any position independently refers to the absence of an additional amino acid residue, or the presence of one or more additional amino acid residues that do not form part of P1, P2 and / or P3.

22. A chimeric polypeptide as defined in any one of claims 1 to 21, and / or A nucleic acid comprising a polynucleotide encoding a chimeric polypeptide as defined in any one of claims 1 to 21, and / or a vector comprising said nucleic acid, and / or a cell comprising said vector.

23. A pharmaceutical composition comprising a cell according to any one of claims 1 to 21, a chimeric polypeptide according to claim 22, a nucleic acid according to claim 22 and / or a vector according to claim 22.

24. A cell according to any one of claims 1 to 21 for use as a medicament.

25. A cell according to any one of claims 1 to 21 for use in treating cancer in a subject.

26. The use a) administering to a subject a population of cells according to any one of claims 1 to 21.

27. The use a) administering to a subject a population of cells according to any one of claims 1 to 21; b) administering to the subject an inhibitor of an inhibitory protease, or a Proteolytic Target Chimeric (Protac) that binds to the Proteolytic Target Chimeric (Protac) binding domain, or a drug that binds the CRBN polypeptide substrate domain to the CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide; and c) increasing or decreasing the concentration of an inhibitor of the inhibitory protease, or a Protac that binds to the proteolytic target chimeric (Protac) binding domain, or the drug that binds the CRBN polypeptide substrate domain to a CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide.

28. 28. The cell of claim 27, wherein the drug in step b) is an IMiD.

29. 29. The cell of claim 28, wherein the IMiD in step b) is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide) and CC-885.

30. The cell of any one of claims 27 to 29, wherein the drug in step c) is an IMiD.

31. 31. The cell of claim 30, wherein the IMiD in step c) is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide) and CC-885.

32. A method of providing a cell according to any one of claims 1 to 21, comprising the step of contacting the cell ex vivo with a nucleic acid or vector according to claim 22.

33. 23. A method for controlling expression of a chimeric polypeptide in a cell, comprising contacting a cell expressing the chimeric polypeptide of claim 22 with an inhibitor of the inhibitory protease, a Proteolytic Target Chimeric (Protac) that binds to the Proteolytic Target Chimeric (Protac) binding domain, or a drug that binds the CRBN polypeptide substrate domain to the CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide, and wherein the cell expressing the chimeric polypeptide is contacted with the inhibitor, Protac, or drug in vitro.

34. 34. The method of claim 33, wherein the drug is an IMiD.

35. 35. The method of claim 34, wherein the IMiD is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide), and CC-885.

36. 1. A method for regulating the cytotoxic activity of T cells and / or NK cells and / or regulating cytokine secretion by T cells and / or NK cells, comprising: a) expressing the chimeric polypeptide of claim 22 in said T cells and / or NK cells ex vivo; and b) contacting the cell in vitro with an inhibitor of the inhibitory protease, a proteolytic target chimeric (Protac) that binds to the Protac binding domain, or a drug that binds the CRBN polypeptide substrate domain to the CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide.

37. 1. A method for regulating the cytotoxic activity of T cells and / or NK cells and / or regulating cytokine secretion by T cells and / or NK cells, comprising: a) expressing the chimeric polypeptide of claim 22 in said T cells and / or NK cells ex vivo; b) contacting the cells in vitro with an inhibitor of the inhibitory protease, a proteolytic target chimeric (Protac) that binds to the Protac binding domain, or a drug that binds the CRBN polypeptide substrate domain to the CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide; and c) increasing or decreasing the concentration of the inhibitor of the inhibitory protease, the Protac, or the CRBN polypeptide substrate domain that binds to the CRBN protein, thereby promoting ubiquitin pathway-mediated degradation of the chimeric polypeptide.

38. 38. The method of claim 36 or 37, wherein the drug in step b) is an IMiD.

39. 39. The method of claim 38, wherein the IMiD in step b) is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide) and CC-885.

40. 40. The method of any one of claims 37 to 39, wherein the drug in step c) is an IMiD.

41. 41. The method of claim 40, wherein the IMiD in step c) is selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122 (avadomide), CC-220 (iberdomide) and CC-885.

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