Method for improving car-t cell effector functions by targeted degradation of glycosylated PD-1

Abstract

The present invention genetically links a small targeting peptide (TP) to E3 ubiquitin ligase β-TrCP and deplete glycosylated, degradation-resistant PD-1 protein from cells by protein knockout technology. The chimeric β-TrCP-TP ubiquitin ligase specifically directs to both glycosylated and non-glycosylated PD-1 protein for destruction. Targeted degradation of endogenous PD-1 by the present invention also promotes marked expansion of CAR T-cells, which is useful in cancer immunotherapy. In one embodiment, TP is a peptide that binds to PD-1. Using a "protein knockout" (PKO)-based targeted protein degradation strategy, the present method selectively depletes glycosylated PD-1. This PKO strategy can be directly incorporated into the CAR T vector, facilitating concurrent delivery of both CAR and PKO, and obviating the need for separate therapeutic regimens of CAR T and anti-PD-1 therapies. The integration of CAR and PKO into a single CAR T vector may significantly enhance the efficacy and durability of CAR T-based immunotherapies.

Description

P-661940-PCMETHOD FOR IMPROVING CAR-T CELL EFFECTOR FUNCTIONS BY TARGETED DEGRADATION OF GLYCOSYLATED PD-1FIELD OF THE INVENTION

[0001] This invention uses protein knockout technology to selectively degrade endogenous PD-1 on CAR T cells. By genetically linking the beta-transducin repeat containing protein (0-TrCP) in P-TrCP-containing E3 ubiquitin ligase with a small fragment of targeting peptide (TP) to bind PD- 1, the resulting chimeric P-TrCP-TP ubiquitin ligase efficiently targets the degradation of PD-1 through the ubiquitin-proteasome machinery.BACKGROUND OF THE INVENTION

[0002] Programmed cell death 1 (PD-1), also known as CD279, is expressed on the cell surface of T cells, B cells, macrophages, and some subsets of dendritic cells upon cell activation and cytokine signaling (S. Simon, N. Labarriere, PD-1 expression on tumor-specific T cells: Friend or foe for immunotherapy? Oncoimmunology 7, el364828 (2017)). Binding of PD-1 to its cognate ligands, PD-L1 (programmed death ligand-1, also known as B7-H1) and PD-L2 (B7-DC), impairs T cell activation, thereby suppressing T cell effector functions, including cell proliferation, cytokine production, and cytolytic activity (G. J. Freeman et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192, 1027-1034 (2000)).

[0003] Glycosylation is a process of attaching oligosaccharides, also known as glycans, to proteins and provides more proteomic diversity than other post-translational modifications (PTMs). Based on the identity of the amino acid bound to a carbohydrate chain, glycosylation can be classified into C-linked, N-linked, O-linked, or S-linked. Particularly, N-linked glycosylation occurs at the asparagine (Asn, N) residue within NXT motifs (Asn-X-Ser / Thr) of extracellular domain or secreted proteins (A. Helenius, M. Aebi, Intracellular functions of N-linked glycans. Science 291, 2364-2369 (2001)). This modification plays important roles in both protein structure and function. For example, N-linked glycosylation has been demonstrated to regulate the interaction between cell surface receptors and their ligands (J. C. Cheung, R. A. Reithmeier, Scanning N-glycosylation mutagenesis of membrane proteins. Methods 41, 451-459 (2007).P-661940-PC

[0004] In addition, N-linked glycans were reported to stabilize lysosome-associated membrane proteins (LAMPs) and protect them from degradation by lysosomal proteases (R. Kundra, S. Kornfeld, Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis. J Biol Chem 274, 31039-31046 (1999)).

[0005] A new frontier in cancer treatment is CAR-T cell therapy, wherein T cells from patients were collected, engineered to express a chimeric antigen receptor (CAR), and reinfused back into the patient to treat the malignancy. CARs are composed of an extracellular antigen recognition domain and intracellular signaling domains. The extracellular antigen recognition domain of the CAR allows CAR-T cells recognize and target specific antigens on cancer cells, while the intracellular signaling domains stimulate T-cell proliferation, cytolysis, and cytokine secretion to eliminate the recognized cancer cells (H. J. Jackson, S. Rafiq, R. J. Brentjens, Driving CAR T- cells forward. Nat Rev Clin Oncol 13, 370-383 (2016)). CAR-T cell therapy has shown promising results for certain blood cancers such as B-cell acute lymphoblastic leukemia, although its efficacy for solid tumors remains less effective (H. J. Jackson, S. Rafiq, R. J. Brentjens, Driving CAR T- cells forward. Nat Rev Clin Oncol 13, 370-383 (2016)).

[0006] Such modest responses in solid tumors may be attributed to the tumor microenvironment (TME). When infused back into patients with solid tumors, CAR-T cells often encounter a suppressive TME, where inhibitory factors act upon the CAR-T cells to suppress their antitumor activities. For example, binding of PD-L1 on ovarian cancer cells to PD-1 on CAR-T cells suppressed T-cell functions (O. O. Yeku, et al. Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment. Sci Rep 7, 10541 (2017)). To this end, blocking PD- 1 / PD-L1 signaling via monoclonal antibodies was found to rescue T-cell activity against cancer cells. Clinical trials for antibody blockade of PD1 / PD-L1 signaling in melanoma (F. S. Hodi et al., Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363, 711 - 723 (2010)) and lung cancer (V. Velcheti et al., Programmed death ligand- 1 expression in nonsmall cell lung cancer. Lab Invest 94, 107-116 (2014)) have resulted in promising new treatment options. Furthermore, disruption of PD-1 / PD-L1 ligation via CRISPR-mediated deletion of PD- L1 on ovarian cancer cells was also found to improve the efficacy of second-generation CAR-T cells in preclinical murine models (L. J. Rupp et al., CRISPR / Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep 7, 737 (2017)).P-661940-PC

[0007] Ubiquitination of target proteins followed by proteasomal degradation serves as a primary mechanism for the post-translational regulation of cellular proteins. Inspired by this mechanism, researchers have developed several strategies for targeted protein degradation (TPD). This emerging field is garnering increasing attention from the academic community and industry, given its potential to therapeutically modulate proteins that are challenging to target with conventional small molecule inhibitors. Currently, the most pervading TPD strategy is PROTAC (proteolysistargeting chimera). This approach utilizes a heterobifunctional small molecule composed of two ligands — one for E3 ubiquitin ligase and another for the target protein — to recruit and direct the proteasome-mediated degradation of the target protein. However, as small molecule inhibitors often cannot target all proteins due to the absence of suitable pockets or grooves for docking, PROTAC’s capacity may also be limited. Thus, there exists a critical need for developing improved therapeutic agents and therapeutically effective methods for the treatment of cancer, including enhancing the efficacy and durability of CAR-T cell therapy in cancers, such as but not limited to solid tumors, for which CAR-T cell therapy is less effective than for blood cancers.SUMMARY OF THE INVENTION

[0008] In one aspect, this disclosure provides a chimeric complex comprising 0-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP- containing SCF E3 ubiquitin ligase.

[0009] In another aspect, this disclosure provides a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding a chimeric complex comprising P-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP-containing SCF E3 ubiquitin ligase, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

[0010] In still another aspect, this disclosure provides a CAR-T cell transduced by a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1P-661940-PC and a second nucleic acid sequence encoding a chimeric complex comprising an engineered 0- TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein 0-TrCP is a 0-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the 0-TrCP in a 0-TrCP-containing SCF E3 ubiquitin ligase, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

[0011] In one aspect, this disclosure provides a method for increasing the ex vivo expansion of CAR T-cells, comprising the steps of transducing T cells with a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding the chimeric complex, wherein the nucleic acid encoding the engineered 0- TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase comprises the nucleic acid sequence set forth in SEQ ID NO: 2; expressing the 0-TrCP-TP E3 ubiquitin ligase thereby selectively depleting glycosylated PD-1 and enhancing the ex vivo expansion of CAR T cells.

[0012] In another aspect, this disclosure provides a method for improving CAR-T cell effector functions, comprising the steps of transducing T cells with a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding an engineered 0-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein 0- TrCP is a 0-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the 0-TrCP in a 0-TrCP-containing SCF E3 ubiquitin ligase, wherein the nucleic acid encoding the 0-TrCP-TP E3 ubiquitin ligase comprises the nucleic acid sequence set forth in SEQ ID NO: 2, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2, thereby selectively depleting glycosylated PD-1, enhancing the ex vivo expansion of CAR T cells, and improving the CAR-T cell effector functions.

[0013] In still another aspect, this disclosure provides a method for in vivo expansion of CAR T- cells in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting 0-TrCP chimeric complex, said complex comprising 0-TrCP E3 ubiquitin ligase fused via a linker to aP-661940-PC targeting peptide (TP) or fragment of the TP, wherein the immune checkpoint protein / receptor is selectively depleted.

[0014] In one aspect, this disclosure provides a method for selectively degrading endogenous PD- 1 in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting P-TrCP chimeric complex, said complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein expression of the P-TrCP chimeric complex selectively degrades endogenous PD-1.

[0015] In another aspect, this disclosure provides a method for enhancing efficacy and anti-tumor activity of a CAR T-cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting P-TrCP chimeric complex, said complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein expression of the CAR and the P-TrCP chimeric complex in the subject enhances the efficacy and anti-tumor activity of the CAR T-cell.

[0016] In one aspect, this disclosure provides a method for deactivating or reversing degradation of a target protein by CAR T-cells in vivo in a subject treated with an anti-tumor CAR T-cell, wherein the target protein is PD-1, the method comprising: inserting a nucleic acid encoding the FKBP 12F36Vdomain at the N terminus of a nucleic acid encoding a PD-1 targeting P-TrCP chimeric complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP to form a PD-1 targeting FKBP12F36V-P-TrCP-TP chimeric complex; transducing the anti-tumor CAR T-cell with a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding the PD-1 targeting FKBP12F36-P-TrCP-TP chimeric complex; administering a therapeutically effective amount of the anti-tumor CAR T-cell comprising the a nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding PD-1 targeting FKBP12F36V- P-TrCP-TP chimeric complex to a subject in need thereof to express the CAR and the PD-1P-661940-PC targeting FKBP12F36V-P-TrCP-TP chimeric complex in the subject, wherein expression of the CAR and the FKBP12F36X-P-TrCP-TP chimeric complex in the subject enhances the efficacy and anti-tumor activity of the CAR T-cell; and administering a degradation tag (dTAG) molecule to the subject to degrade the PD-1 targeting P-TrCP-TP chimeric complex, thereby deactivating or reversing degradation of PD-1 to alleviate or reduce adverse effects of the degradation of PD-1.

[0017] In another aspect, this disclosure provides a retroviral CAR construct comprising a nucleic acid encoding an anti-tumor CAR, a nucleic acid encoding P-TrCP-TP chimera, and a nucleic acid encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP-TP chimera.

[0018] These and other aspects of the invention will be appreciated from the ensuing detailed description, examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating certain embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to one skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figures 1A-1I show N-linked glycosylation stabilizes PD-1 protein by preventing GSK3P-induced degradation. (Fig. 1A) Schematic diagram of GSK30 phosphorylation motif on PD-1. (Fig. IB) Increasing amounts of FLAG-tagged GSK3P were co-expressed with Myc-tagged PD-1 WT in HEK293T cells. SDS lysis buffer was used to lyse cells. PD-1 protein levels were analyzed by Western blot. (Fig. 1C) PD-1 WT was overexpressed in HEK293T cells. Cell lysates treated with N-linked glycosidase PNGase F or O-linked glycosidase were analyzed by Western blot. Glycosylated PD-1 (black circle), unglycosylated PD-1 (black arrow). (Fig. ID) HEK293T cells overexpressing PD-1 WT were treated with the N-linked glycosylation inhibitor tunicamycin (TM, 5ug / ml) for 12 or 16 hours. PD-1 protein levels were assessed by Western blot. Glycosylated PD-1 (black circle), unglycosylated PD-1 (black arrow). (Fig. IE) Schematic diagram of the putative N-linked glycosylation sites on PD-1 protein. Full-length PD-1 is separated into an extracellular domain (ECD) and an intracellular domain (ICD) by the transmembrane domain (TM). Four N-linked glycosylation sites (NXT motifs) were identified in the ECD (red). SP, signal peptide. (Fig. IF) PD-1 WT, NQ mutants (left panel) and random N to Q mutants (right panel)P-661940-PC were overexpressed in HEK293T cells. 2NQ, N49Q / N58Q; 3NQ, N49Q / N58Q / N74Q; 4NQ, N49Q / N58Q / N74Q / N116Q. (Fig. 1G) SK-OV-3 cells overexpressing PD-1 WT were treated with DMSO or 100 mg / ml cycloheximide (CHX) for the indicated times. PD-1 protein levels were analyzed by Western blot under both normal and extended exposure time. Glycosylated PD-1 (black circle), unglycosylated PD-1 (black arrow). (Fig. 1H) Increasing amounts of FLAG-tagged GSK3P were co-expressed with glycosylation-deficient PD-1 4NQ mutant. Protein levels of PD- 1 4NQ were analyzed by Western blot. (Fig. II) Myc-tagged PD-1 WT or NQ mutants were coexpressed with FLAG-tagged GSK3P in HEK293T cells. GSK3P proteins were pulled down by anti-FLAG (M2) antibody from cell lysates. FLAG immunoprecipitates and the input fraction (10%) were assessed for PD-1 and GSK3P expression.

[0020] Figures 2A-2B show N-linked glycosylated PD-1 is predominantly membrane localized. (Fig. 2A) PD-1 WT and different NQ mutants were transfected into SK-OV-3 cells and assessed by fractionation and immunofluorescence of PD-1 protein levels. (Fig. 2B) PD-1 WT, PD-1 N49Q and PD-1 4NQ were transfected in SK-OV-3 cells. Their intracellular localization was assessed by immunofluorescent staining. PD-1 protein (green), membrane marker NK-ATPase (red). Nucleus (blue).

[0021] Figures 3A-3B show N-linked glycosylation interferes with PD-1 and PD-L1 interaction. (Fig. 3A) Myc-tagged PD-1 WT or N to Q mutants were co-expressed with FLAG-tagged PD-L1 in HEK293T cells. PD-L1 proteins were pulled down by anti-FLAG (M2) antibody from cell lysates and analyzed along with the input fraction (10%). (Fig. 3B) Myc-tagged PD-1 WT was coexpressed with FLAG-tagged PD-L1 in HEK293T cells. N-linked glycosylation inhibitor tunicamycin was added at 5 pg / ml for the indicated time before harvest. Anti-Myc tag antibody was used to pull down PD-1 proteins from cell lysates. Myc tag immunoprecipitates and the input fraction (10%) were assessed for PD-1 and PD-L1 expression.

[0022] Figures 4A-4F show engineered 0-TrCP chimera targets glycosylated PD-1 for degradation. (Fig. 4A) Schematic diagram of Protein Knockout Technology (PKO). F-box protein in SCF E3 ubiquitin ligase complex (left) was substituted by the 0-TrCP chimera (right) containing the targeting peptide (TP) specific for binding of targets of interested (neo-substrate). (Fig. 4B) Schematic diagram of PD-1 (a) and SHP-2 phosphatase (b). ITIM and ITSM motifs in the intracellular domain (ICD) of PD-1 are binding sites for SH2 (C) and SH2 (N) domains of SHP2, respectively. (Fig. 4C) Structures of three different 0-TrCP expression vectors used in this study,P-661940-PC a. full length of P-TrCP WT with no linker sequence; b. full length of 0-TrCP WT with 10GS sequence as linker; c. truncated P-TrCP without WD40 domain with 10GS sequence as linker. (Fig. 4D) Myc-tagged PD-1 WT was co-expressed with various FLAG-tagged P-TrCP chimeras in HEK293T cells. PD-1 protein levels were analyzed by Western blot. (Fig. 4E) PD-1 WT was coexpressed with increasing amounts of FLAG-tagged chimeric P-TrCP.10GS.SH2(C) (left panel) or FLAG-tagged P-TrCP(AWD). 10GS.SH2(C) (right panel) in HEK293T cells. PD-1 protein levels were analyzed by Western blot. Glycosylated PD-1 (black circle) (Fig. 4F) Myc-tagged PD- 1 WT was co-expressed with FLAG-tagged P-TrCP.10GS.SH2(C) or P- TrCP(AWD).10GS.SH2(C) in HEK293T cells. P-TrCP chimeras were pulled down by anti-FLAG (M2) antibody. PD-1 WT in FLAG immunoprecipitates and the input fraction (10%) were assessed by Western blot.

[0023] Figures 5A-5H show P-TrCP chimera promotes ex vivo expansion of CAR-T cells. (Fig. 5A) Retroviral particles from P-TrCP chimeras cloned into the pBMN.GFP vector was transduced into Jurkat cells through spinoculation and assessed for downregulation of PD-1. (Fig. 5B) Schematic diagram of empty CAR construct (upper) and CAR-P2A-P-TrCP chimera (lower) used in this study. (Fig. 5C) Retroviral CAR-P2A-P-TrCP chimera constructs were transduced into primary T cells from three different donors to generate CAR-T cells. Western blot was performed to detect endogenous PD-1 expression in CAR T-cells. (Fig. 5D) Flow cytometric analysis of CAR expression (left panel) and transduction efficiency (right panel) in basal CAR-T group and P-TrCP chimera-containing CAR-T group, ns, no significance. (Fig. 5E) Flow cytometric analysis was conducted to determine the percentage of PD-1 positive cells in basal CAR-T cells and P-TrCP chimera-containing CAR-T cells. **P < 0.01. (Fig. 5F) Flow cytometric analysis was performed to measure PD-1 expression levels in basal CAR-T group and 0-TrCP chimera-containing CAR-T group. **P < 0.01. (Fig. 5G) (Fig. 5H) Ex vivo proliferation assay was conducted for both basal CAR-T cells and 0-TrCP chimera-containing CAR-T cells by co-culturing with PD-L1 -stably expressing SK-OV-3 cells. Flow cytometric analysis was performed to measure PD-1 expression levels (Fig. 5G) and to enumerate cell numbers (Fig. 5H) in both groups at indicated time point, ns, no significance. **P < 0.01.

[0024] Figure 6 shows protein sequence alignment for different origins ofPD-1. Highly conserved N49, N74 and Nil6 were emphasized by red frame. Moderately conserved N58 was labeled by blue frame.P-661940-PC

[0025] Figures 7A-7B show post-translational N-glycosylation of PD-1 shields it from GSK30- mediated degradation and maintains its presence on the surfaces of CAR-T cells, reinforcing the PD-1 / PD-L1 immune checkpoint (“on”) and suppressing CAR-T cell proliferation compared to the presently provided “protein knockout” (PKO)-based targeted protein degradation strategy in which both CAR and PKO are directly incorporated into a CAR-T vector, which degrades endogenous PD-1 and significantly enhances the antitumor efficacy and durability / proliferation of CAR-T cell immunotherapy and immune checkpoint inhibition (“off’).

[0026] Figures 8A-8H show N-linked glycosylation stabilizes PD-1 protein by preventing GSK3b-induced degradation. (Fig. 8A) Schematic diagram of GSK30 phosphorylation motif on PD-1. (Fig. 8B) Increasing amounts of FLAG-tagged GSK3 were co-expressed with Myc-tagged PD-1 WT in HEK293T cells. SDS lysis buffer was used to lyse cells. PD-1 protein levels were analyzed by Western blot. (Fig. 8C) Increasing amounts of FLAG-tagged GSK3P were coexpressed with glycosylation-deficient PD-1 4NQ mutant. Protein levels of PD-1 4NQ were analyzed by Western blot. (Fig. 8D) Jurkat T lymphocytes were activated by ImmunoCult™ Human CD3 / CD28 T Cell Activator for 3 days. After that, FLAG-tagged GSK3b was transfected into activated Jurkat cells by nucleofection with Lonza SE Cell Line 4D- Nucleofector™ X Kit according to manufacturer’s protocol. Program CL-120 was used for nucleofection. Endogenous glycosylated PD-1 and unglycosylated PD-1 were analyzed by Western blot. Glycosylated PD-1 (black circle), unglycosylated PD-1 (black arrow). (Fig. 8E) Myc-tagged PD-1 WT or NQ mutants were co-expressed with FLAG-tagged GSK30 in HEK293T cells. GSK30 proteins were pulled down by anti-FLAG (M2) antibody from cell lysates. FLAG immunoprecipitates and the input fraction (10%) were assessed for PD-1 and GSK30 expression. (Fig. 8F) PD-1 WT and phosphorylation-deficient mutants were overexpressed with or without GSK3 P in HEK293T cells for 48 hours. After that, cells were harvested and lysed by SDS buffer. PD-1 protein levels in cell lysates were analyzed by Western blot. (Fig. 8G) PD-1 phosphorylation / glycosylation doubledeficient mutants were overexpressed with or without GSK3b in HEK293T cells. After lysis by SDS buffer, PD-1 protein levels were measured by Western blot. (Fig. 8H) PD-1 phosphorylation deficient mutants and NQ mutants were overexpressed in HEK293T cells. PD-1 protein levels in cell lysate were analyzed by Western blot.

[0027] Figures 9A-9F show PD-1 protein levels assessments, N-linked glycosylation sites on PD- 1 protein, and N-linked glycosylation sites on PD-1. Fig. 9A shows PD-1 WT was overexpressedP-661940-PC in HEK293T cells. Cell lysates treated with N-linked glycosidase PNGase F or O-linked glycosidase were analyzed by Western blot. Glycosylated PD-1 (black circle), unglycosylated PD- 1 (black arrow). Fig. 9B shows HEK293T cells overexpressing PD-1 WT were treated with the N- linked glycosylation inhibitor tunicamycin (TM, 5ug / ml) for 12 or 16 hours. PD-1 protein levels were assessed by Western blot. Glycosylated PD-1 (black circle), unglycosylated PD-1 (black arrow). Fig. 9C shows a schematic diagram of the putative N-linked glycosylation sites on PD-1 protein. Full-length PD-1 is separated into an extracellular domain (ECD) and an intracellular domain (ICD) by the transmembrane domain (TM). Four N-linked glycosylation sites (NXT motifs) were identified in the ECD (red). SP, signal peptide. Fig. 9D shows a protein sequence alignment for PD- 1 from different species. Highly conserved N49, N74 and N116 were highlighted by red frame. Moderately conserved N58 was labeled by blue frame. Fig. 9E shows PD-1 WT, NQ mutants (left panel) and random N to Q mutants (right panel) were overexpressed in HEK293T cells. Cell lysates were analyzed by Western blot. 2NQ, N49Q / N58Q; 3NQ, N49Q / N58Q / N74Q; 4NQ, N49Q / N58Q / N74Q / N116Q. Fig. 9F shows SK-OV-3 cells overexpressing PD-1 WT were treated with DMSO or 100 mg / ml cycloheximide (CHX) for the indicated times. PD-1 protein levels were assessed by Western blot under both normal and extended exposure time. Glycosylated PD-1 (black circle), unglycosylated PD-1 (black arrow).

[0028] Figures 10A-10B show N-linked glycosylation interferes with PD-1 and PD-L1 interaction. Fig. 10A shows Myc-tagged PD-1 WT or N to Q mutants were coexpressed with FLAG-tagged PD-L1 in HEK293T cells. PD-L1 proteins were pulled down by anti-FLAG (M2) antibody from cell lysates and analyzed along with the input fraction (10%). Fig. 10B shows Myc- tagged PD-1 WT was co-expressed with FLAG-tagged PD-L1 in HEK293T cells. N-linked glycosylation inhibitor tunicamycin was added at 5 ug / ml for the indicated time before harvest. Anti-Myc tag antibody was used to pull down PD-1 proteins from cell lysates. Myc tag immunoprecipitates and the input fraction (10%) were assessed for PD-1 and PD-L1 expression.

[0029] Figures 11A-11B show PD-1 WT and different NQ mutants were transfected into SK- OV-3 cells and assessed by fractionation and immunofluorescence of PD-1 protein levels (Fig. 11A). Fig. 11B shows PD-1 WT, PD-1 N49Q and PD-1 4NQ were transfected in SK-OV-3 cells. Their intracellular localization was assessed by immunofluorescent staining. PD-1 protein (green), membrane marker NK-ATPase (red). Nucleus (blue).P-661940-PC

[0030] Figures 12A-12G show targeting glycosylated, degradation-resistant PD-1 via engineered P-TrCP chimeric ligases. Fig. 12A a schematic diagram of Protein Knockout Technology (PKO). F-box protein in SCF E3 ubiquitin ligase complex (left) was substituted by the P-TrCP chimera (right) containing the targeting peptide (TP) specific for binding of targets of interested (neosubstrate). Fig. 12B a schematic diagram of PD-1 and SHP-2 phosphatase. ITIM and ITSM motifs in the intracellular domain (ICD) of PD-1 are binding sites for SH2 (N) and SH2 (C) domains of SHP2, respectively. Fig. 12C shows structures of three different 0-TrCP expression vectors used in this study. Full length of -TrCP WT with no linker sequence; Full length of 0-TrCP WT with 10GS sequence as linker; Truncated 0-TrCP without WD40 domain with 10GS sequence as linker. Fig. 12D shows Myc-tagged PD-1 WT was co-expressed with various FLAG-tagged P-TrCP chimeras in HEK293T cells. PD-1 protein levels were analyzed by Western blot. (12E) PD-1 WT was co-expressed with increasing amounts of FLAG-tagged chimeric P-TrCP.10GS.SH2(C) (upper panel) or FLAG-tagged P-TrCP(A WD).10GS.SH2(C) (lower panel) in HEK293T cells. PD-1 protein levels were analyzed by Western blot. Fig. 12F shows PD-1 4NQ mutant was coexpressed with increasing amounts of FL AG-tagged chimeric P-TrCP.10GS.SH2(C) in HEK293T cells. Protein levels of PD-1 4NQ were analyzed by Western blot. Fig. 12G shows Myc-tagged PD-1 WT was co-expressed with FLAG-tagged P-TrCP.10GS.SH2(C) or P- TrCP(AWD).10GS.SH2(C) in HEK293T cells. P-TrCP chimeras were pulled down by anti -FLAG (M2) antibody. PD-1 WT in FLAG immunoprecipitates and the input fraction (10%) were assessed by Western blot.

[0031] Figures 13A-13B show Myc-tagged PD-1 WT or NQ mutants were overexpressed in HEK293T cells. PD-Ll-Fc was used to pull down PD-1 proteins from cell lysate (Fig. 13A). PD- 1 protein levels in PD-Ll-Fc precipitates and input fraction (10%) were analyzed by Western blot. Following normalization of PD-1 input and pull-down signals, relative binding efficiencies for all single and combinatorial N-to-Q mutants were calculated. Fig. 13B shows the data in histogram are representative of three independent experiments.

[0032] Figures 14A-14H show P-TrCP chimera promotes ex vivo expansion of CAR-T cells. Fig. 14A shows retroviral particles from P-TrCP chimeras cloned into the pBMN.GFP vector was transduced into Jurkat cells through spinoculation and assessed for downregulation of PD-1. Fig. 14B shows a schematic diagram of empty CAR construct (upper) and CAR-P2A-P-TrCP chimera (lower) used in this study. Figs. 14C(a)-14C(c) shows retroviral CAR-P2A-P-TrCP chimeraP-661940-PC constructs were transduced into primary T cells from three different donors to generate CAR-T cells. Western blot was performed to detect endogenous PD-1 expression in CAR T-cells. Fig. 14D shows a histogram graph of flow cytometric analysis of CAR expression (left panel) and transduction efficiency (right panel) in basal CAR-T group and 0-TrCP chimera-containing CAR- T group, ns, no significance. Fig. 14E shows a histogram graph of flow cytometric analysis was conducted to determine the percentage of PD-1 positive cells in basal CAR-T cells and 0-TrCP chimera-containing CAR-T cells. **P < 0.01. Fig. 14F shows flow cytometric analysis that was performed to measure PD-1 expression levels in PD-1 positive cells from basal CAR-T group and 0-TrCP chimera-containing CAR-T group. **P < 0.01. (Fig. 14G) (14H) Ex vivo proliferation assay was conducted for both basal CAR-T cells and P-TrCP chimera-containing CAR-T cells by co-culturing with PD-L1 -stably expressing SK-OV-3 cells. Flow cytometric analysis was performed to measure PD-1 expression levels (Fig. 14G) and to enumerate cell numbers (Fig. 14H) in both groups at indicated time point, ns, no significance. **P < 0.01.

[0033] Figures 15A-15F show contingency Control: dTAG-mediated shutdown of PKO activity. Fig. 15A shows a schematic diagram of dTAG molecule-mediated degradation of FKBP12F36V- fused PKO construct. Fig. 15B shows PKO constructs and FKBP12F36V-fused PKO constructs were overexpressed in HEK293T cells. Cell lysates were assessed by Western blot. Fig. 15C shows FKBP12F36V-fused PKO constructs were overexpressed in HEK293T cells for 36 hours. After that, cells were treated with dTAGV-1, dTAG-7 and dTAG-13 at indicated concentration for 24 hours. Cells lysates were harvested and analyzed by Western blot. Fig. 15D shows FKBP12F36V-fused PKO constructs were overexpressed in HEK293T cells for 36 hours. Then cells were treated with 10 mM of three different dTAG molecules, dTAGV-1, dTAG-7 and dTAG-13 for indicated time. Cells were lysed by SDS buffer and analyzed by Western blot. Fig. 15E shows FKBP12F36V-fused PKO constructs were overexpressed in SH-SY-5Y neuroblastoma cells for 48 hours. Then cells were treated with 10 mM of dTAGV-1, dTAG-7 and dTAG-13 for indicated time. Cells were lysed by SDS buffer and analyzed by Western blot. Fig. 15F shows FKBP12F36V-fused PKO constructs were coexpressed with PD-1 WT in HEK293T cells for 36 hours. Then cells were treated with 1 mM of dTAGV-1, dTAG-7 and 0.1 mM dTAG-13 for 24 hours. After that, cells were harvested and analyzed by Western blot.

[0034] Figures 16A-16H show an exemplary engineered CAR-PKO construct of the present invention and the nucleic acid sequence encoding the CAR (SEQ ID NO: 1). As shown in aP-661940-PC schematic (Fig. 16A), the whole construct was composed of a 4H1128Z CAR, then followed by a P2A sequence linking upstream CAR component and downstream P-TrCP chimera. The nucleic acid sequence of the 4H1128Z CAR is shown (Fig. 16B) in which the gray part is the extracellular (EC) domain of the CAR (1 - 933 bp), the white part is the transmembrane (TM) domain of the CAR (934 - 1014), the green part is the CD28 domain of the intracellular (IC) domain of the CAR (1015 - 1137 bp), and the pink part is the CD3z domain of the intracellular (IC) domain of the CAR (1138 - 1473 bp). In an embodiment, the CAR-PKO construct comprises the P-TrCP (full length) chimera nucleic acid sequence (SEQ ID NO: 2). In an embodiment, the CAR-PKO construct comprises the P-TrCP (AWD) chimera nucleic acid sequence (SEQ ID NO: 3).

[0035] Figure 17 shows the nucleic acid sequence of the engineered P-TrCP (full length) chimera sequence (2100 bp) (SEQ ID NO:2) of the present invention.

[0036] Figure 18 shows the nucleic acid sequence of the engineered P-TrCP (AWD) chimera sequence (1170 bp) (SEQ ID NO:3) of the present invention comprising the gray part: P-TrCP (1- 1707), the white part: 10GS linker (1708-1791), the blue part: SH2 (C) targeting peptide (1792- 2100). In an embodiment, P-TrCP (full length) chimera and the P-TrCP (AWD) chimera, respectively, comprise a nucleic acid sequence encoding the targeting peptide SH2(C) sequence (309 bp) (SEQ ID NO: 15). In an embodiment, P-TrCP (full length) chimera and the P-TrCP (AWD) chimera, respectively, comprise a nucleic acid sequence encoding the targeting peptide SH2 (N) sequence (306 bp) (SEQ ID NO: 16).

[0037] Figure 19 shows the nucleic acid sequence encoding the 10GS linker sequence (60 bp) (SEQ ID NO: 10) used in the engineered P-TrCP (full length) chimera and the engineered P-TrCP (AWD) chimera.

[0038] Figure 20 shows the nucleic acid sequence encoding PD-1 (867 bp) (SEQ ID NO: 11).

[0039] Figure 21 shows the nucleic acid sequence encoding the PD-1 extracellular domain (also called “ECD” or “EC” herein) (70 - 501 bp) (SEQ ID NO: 12).

[0040] Figure 22 shows the nucleic acid sequence encoding the PD-1 transmembrane domain (“TM” herein) (502 - 570 bp) (SEQ ID NO: 13).

[0041] Figure 23 shows the nucleic acid sequence encoding the PD-1 intracellular domain (“ICD” or “IC”) (“herein) (571- 867 bp) (SEQ ID NO: 14).

[0042] Figure 24 shows the nucleic acid sequence encoding the targeting peptide SH2(C) sequence (309 bp) (SEQ ID NO: 15).P-661940-PC

[0043] Figure 25 shows the nucleic acid sequence encoding the targeting peptide SH2 (N) sequence (306 bp) (SEQ ID NO: 16).

[0044] Figure 26 shows the nucleic acid sequence encoding the FKBP12J’3oVsequence (327 bp) (SEQ ID NO: 17).

[0045] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.DETAILED DESCRIPTION

[0046] Programmed cell death-1 (PD-1) is expressed on the cell surface of immune effector cells, such as T and NK cells following activation. Interaction between PD-1 and its cognate ligand, programmed death ligand-1 (PD-L1) inhibits T-cell effector functions, including proliferation, cytokine production and cytolytic activity, leading to immune escape. The inventors have discovered a critical role of N-linked glycosylation in regulating stability and subcellular localization of PD-1 and identified N-linked glycosylation sites on PD-1. The disruption of glycosylation on these sites significantly decreased PD-1 protein stability resulting in cytosolic localization of PD-1. The inventors have also shown that N-linked glycosylation of PD-1 stabilizes membrane localization by inhibiting the binding of glycogen synthase kinase 3b (GSK3P), thereby blocking phosphorylation-induced degradation of PD-1 by GSK3P andE3 ubiquitin ligase P-TrCP.

[0047] The present invention addresses the need for providing improved compositions, such as CAR-T cells, and therapeutic methods comprising administering CAR-T cells to treat cancers and tumors by targeted degradation of target proteins / receptors on CAR-T cells, including degradation of glycosylated PD-1 with protein knockout technology that improves CAR T-cell effector functions by selectively degrading endogenous PD-1, and enhancing efficacy and anti -tumor activity of a CAR T-cell in a subject. The present invention also provides dTAG-mediated shutdown of PKO activity to rapidly mitigate potential adverse effects from targeted protein degradation, including PD-1 degradation.

[0048] The present invention uses protein knockout technology to selectively degrade endogenous PD-1 on CAR-T cells. By genetically linking E3 ubiquitin ligase P-TrCP with a small fragment ofP-661940-PC targeting peptide (TP) to bind proteins of interest (e.g., PD-1), the resulting chimeric 0-TrCP-TP ubiquitin ligase efficiently targets the degradation of both glycosylated and non-glycosylated PD- 1 protein for destruction through the ubiquitin-proteasome machinery.

[0049] In one aspect, the invention is directed to a chimeric complex of P-TrCP-TP SCF (Skpl- Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP-containing SCF E3 ubiquitin ligase.

[0050] In SCF (Skpl-Cullinl -F-box) E3 ubiquitin ligases complex, F-box protein such as P-TrCP is responsible for substrate recognition and binding. By directly fusing the F-box protein with a targeting peptide (TP), a small peptide capable of binding to the substrate of interest, the resulting chimeric F-box protein specifically recognizes and binds to the intended substrates, and then target the substrates for degradation through the ubiquitin-proteasome system. Since glycosylation on PD-1 could prevent the binding of GSK3P, the non-glycosylated intracellular domain of PD-1 is a candidate site for chimeric F-box protein.

[0051] Tyrosine phosphatase SHP-2 is a major mediator for the inhibitory activity of PD-1. Following recruitment to the cytoplasmic domain of PD-1, SHP-2 dephosphorylates the downstream molecule of TCR / CD28 signaling and transmits inhibitory signals. Two SH2 domains at N-terminal of SHP-2, SH2 (N) and SH2 (C), are required for complete phosphatase activity. In one embodiment, TP is SH2 (C) domain of Tyrosine phosphatase SHP2. In one embodiment, TP is SH2 (N) domain of Tyrosine phosphatase SHP2. In one embodiment, TP is SH2 (C) domain plus SH2 (N) domain of Tyrosine phosphatase SHP2.

[0052] In one aspect, the invention is directed to a vector comprising a first nucleic acid sequence encoding a CAR (chimeric antigen receptor) and a second nucleic acid sequence encoding the chimeric complex as described above.

[0053] In one aspect, the invention is directed to CAR-T cells transduced by the above vector. CAR T cells co-expressing this chimera dramatically decreased endogenous PD-1 expression when as compared to unmodified CAR T cells.

[0054] In one aspect, this disclosure provides a chimeric complex comprising 0-TrCP-TP SCF (Skpl-Cullinl -F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP- containing SCF E3 ubiquitin ligase. In an embodiment, the TP is SH2 (C) and / or SH2 (N) domainsP-661940-PC of tyrosine phosphatase SHP2. In an embodiment, the TP is SH2 (C) (SEQ ID NO: 15) and / or SH2 (N) domains (SEQ ID NO: 16) of Tyrosine phosphatase SHP2. In an embodiment, the P-TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:3. In an embodiment, the P-TrCP chimeric complex is a PD-1 targeting P-TrCP chimeric complex encoded by SEQ ID NO: 2 or SEQ IDN0:3. In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the P-TrCP chimeric further comprises a nucleic acid encoding a self-cleaving 2 A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.

[0055] In another aspect, this disclosure provides a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding a chimeric complex comprising P-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP-containing SCF E3 ubiquitin ligase, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2. In an embodiment, the TP is SH2 (C) and / or SH2 (N) domains of Tyrosine phosphatase SHP2. In an embodiment, the TP is SH2 (C) (SEQ ID NO: 15) and / or SH2 (N) domains (SEQ ID NO: 16) of Tyrosine phosphatase SHP2. In an embodiment, the chimeric complex is a PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P- TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the chimeric complex is a PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P- TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the P-TrCP chimeric further comprises a nucleic acid encoding a self-cleaving 2A peptide derived from porcineP-661940-PC teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.

[0056] In still another aspect, this disclosure provides a CAR-T cells transduced by a vector a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding a chimeric complex comprising P-TrCP- TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP-containing SCF E3 ubiquitin ligase, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2. In an embodiment, the TP is SH2 (C) and / or SH2 (N) domains of tyrosine phosphatase SHP2. In an embodiment, the TP is SH2 (C) (SEQ ID NO: 15) and / or SH2 (N) domains (SEQ ID NO: 16) of Tyrosine phosphatase SHP2. In an embodiment, the chimeric complex is a PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3).

[0057] In one aspect, this disclosure provides a method for increasing the ex vivo expansion of CAR T-cells, comprising the steps of transducing T cells with a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding the chimeric complex, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2; expressing the CAR thereby selectively depleting glycosylated PD-1 and enhancing the ex vivo expansion of CAR T cells. In an embodiment, the TP is SH2 (C) and / or SH2 (N) domains of Tyrosine phosphatase SHP2. In an embodiment, the TP is SH2 (C) (SEQ ID NO: 15) and / or SH2 (N) domains (SEQ ID NO: 16) of tyrosine phosphatase SHP2. In an embodiment, the chimeric complex is a PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the P-TrCP chimeric further comprises a nucleic acidP-661940-PC encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the -TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.

[0058] In another aspect, this disclosure provides a method for improving CAR-T cell effector functions, comprising the steps of transducing T cells with a vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding a chimeric complex comprising [3-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targeting peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP-containing SCF E3 ubiquitin ligase, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2, thereby selectively depleting glycosylated PD- 1, enhancing the ex vivo expansion of CAR T cells, and improving the CAR-T cell effector functions. In an embodiment, the TP is SH2 (C) and / or SH2 (N) domains of Tyrosine phosphatase SHP2. In an embodiment, the TP is SH2 (C) (SEQ ID NO: 15) and / or SH2 (N) domains (SEQ ID NO: 16) of Tyrosine phosphatase SHP2. In an embodiment, the chimeric complex is a PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the 0-TrCP chimeric further comprises a nucleic acid encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the 0-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.

[0059] In still another aspect, this disclosure provides a method for in vivo expansion of CAR T- cells in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting P-TrCPP-661940-PC chimeric complex, said complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein the immune checkpoint protein / receptor is selectively depleted. In an embodiment, the immune checkpoint protein / receptor-targeting 0-TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:3. In an embodiment, the chimeric antigen receptor (CAR) is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the linker is 10GS, and the 10GS encoded by a nucleic acid sequence set forth in SEQ ID NO: 10. In an embodiment, the TP or fragment of the TP binds to the immune checkpoint immune checkpoint protein / receptor, and the immune checkpoint immune checkpoint protein / receptor is PD-1, and the PD-1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the endogenous PD-1 is on CAR-T cells and is glycosylated. In an embodiment, the endogenous PD-1 on CAR-T cells and is non-glycosylated. In a particular embodiment, the TP is a SH2 (C) domain of tyrosine phosphatase SHP2, and the SH2 (C) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the TP is a SH2 (N) domain of tyrosine phosphatase SHP2, and the SH2 (N) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 16. In an embodiment, the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the CAR comprises an extracellular (EC) antigen recognition domain, a transmembrane (TM) domain and an intracellular (IC) signaling domain. In an embodiment, the CAR comprises an extracellular (EC) antigen recognition domain (SEQ ID NO:4), a transmembrane (TM) domain (SEQ ID NO:5) and an intracellular (IC) signaling domain (SEQ ID NO: 6). In an embodiment, the chimeric complex is a PD-1 targeting P-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12r36Vdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting 0-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the P- TrCP chimeric further comprises a nucleic acid encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.P-661940-PC

[0060] In one aspect, this disclosure provides a method for selectively degrading endogenous PD- 1 in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting p-TrCP chimeric complex, said complex comprising 0-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein expression of the 0-TrCP chimeric complex selectively degrades endogenous PD-1. In an embodiment, the immune checkpoint protein / receptor-targeting 0-TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1. In an embodiment, the chimeric antigen receptor (CAR) is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the linker is 10GS, and the 10GS encoded by a nucleic acid sequence set forth in SEQ ID NO: 10. In an embodiment, the TP or fragment of the TP binds to the immune checkpoint immune checkpoint protein / receptor, and the immune checkpoint immune checkpoint protein / receptor is PD-1, and the PD-1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the endogenous PD- 1 is on CAR-T cells and is glycosylated. In an embodiment, the endogenous PD-1 on CAR-T cells and is non-glycosylated. In certain embodiments, the TP is a SH2 (C) domain of tyrosine phosphatase SHP2, and the SH2 (C) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15. In an embodiment, the TP is a SH2 (N) domain of tyrosine phosphatase SHP2, and the SH2 (N) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 16. In a particular embodiment, the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 5 and a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 6. In an embodiment, the CAR comprises an extracellular (EC) antigen recognition domain, a transmembrane (TM) domain and an intracellular (IC) signaling domain. In an embodiment, the CAR comprises an extracellular (EC) antigen recognition domain (SEQ ID NO:4), a transmembrane (TM) domain (SEQ ID NO:5) and an intracellular (IC) signaling domain (SEQ ID NO: 6). In an embodiment, the chimeric complex is a PD-1 targeting 0-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NOG). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36domain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting - TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NOG). In an embodiment, the 0-TrCP chimericP-661940-PC further comprises a nucleic acid encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.

[0061] In another aspect, this disclosure provides a method for enhancing efficacy and anti-tumor activity of a CAR T-cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting P-TrCP chimeric complex, said complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein expression of the CAR and the P-TrCP chimeric complex in the subject enhances the efficacy and anti-tumor activity of the CAR T-cell. In an embodiment, the immune checkpoint protein / receptor- targeting P-TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1. In an embodiment, the chimeric antigen receptor (CAR) is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the linker is 10GS, and the 10GS encoded by a nucleic acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the TP or fragment of the TP binds to the immune checkpoint immune checkpoint protein / receptor, and the immune checkpoint immune checkpoint protein / receptor is PD-1, and the PD-1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the 0-TrCP chimeric complex selectively degrades endogenous PD-1. In an embodiment, the endogenous PD-1 is on CAR-T cells and is glycosylated. In some embodiments, the endogenous PD-1 on CAR-T cells and is nonglycosylated. In a particular embodiment, the TP is a SH2 (C) domain of tyrosine phosphatase SHP2, and the SH2 (C) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15. In an embodiment, the TP is a SH2 (N) domain of tyrosine phosphatase SHP2, and the SH2 (N) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 16. In a particular embodiment, the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 5 and a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 6. In an embodiment, the CAR comprises an extracellular (EC) antigen recognition domain, a transmembrane (TM) domain and an intracellular (IC) signaling domain. In an embodiment, the CAR comprises an extracellular (EC) antigenP-661940-PC recognition domain (SEQ ID NO:4), a transmembrane (TM) domain (SEQ ID NO:5) and an intracellular (IC) signaling domain (SEQ ID NO: 6). In an embodiment, the chimeric complex is a PD-1 targeting 0-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In some embodiments, the chimeric complex further comprises a nucleic acid encoding a FKBP12F36Xdomain (SEQ ID NO: 17) at the N terminus of the nucleic acid encoding the PD-1 targeting P- TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3). In an embodiment, the P-TrCP chimeric further comprises a nucleic acid encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9. In one aspect, this disclosure provides a method for deactivating or reversing degradation of a target protein by CAR T-cells in vivo in a subject treated with an anti-tumor CAR T-cell, wherein the target protein is PD-1, the method comprising: (a) inserting a nucleic acid encoding the FKBP12F36Vdomain at the N terminus of a nucleic acid encoding a PD-1 targeting P-TrCP chimeric complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP to form a PD-1 targeting FKBP12F36V-P-TrCP chimeric complex; (b) transducing the anti-tumor CAR T-cell with a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding the PD-1 targeting FKBP12F36V-P-TrCP chimeric complex; (c) administering a therapeutically effective amount of the anti-tumor CAR T-cell comprising the a nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding PD-1 targeting FKBP12F36V- P-TrCP chimeric complex to a subject in need thereof to express the CAR and the PD-1 targeting FKBP12F36V-P-TrCP chimeric complex in the subject, wherein expression of the CAR and the FKBP12F36V-P-TrCP chimeric complex in the subject enhances the efficacy and anti-tumor activity of the CAR T-cell; and (d) administering a degradation tag (dTAG) molecule to the subject to degrade the PD-1 targeting P-TrCP chimeric complex, thereby deactivating or reversing degradation of PD-1 to alleviate or reduce adverse effects of the degradation of PD-1. In a particular embodiment, the dTAG molecule is dTAG-7, dTAG-13, or dTAGV-1. dTAG-7 and dTAG-13. The degradation tag (dTAG) molecule may be administered to the subject in an amount effective to degrade the PD-1 targeting P-TrCP chimeric complex, thereby deactivating or reversing degradation of PD-1 to alleviate or reduce adverse effects of the degradation of PD-1. InP-661940-PC an embodiment, administration of dTAG-7, a degrader for mutant FKBP12F36Vfusion proteins comprises a ligand selective for F36V single-point mutated FKBP12, a linker and a cereblon- binding ligand. Administration of dTAG-7 is known to induce a rapid, reversible and selective degradation of FKBP12F36Vfusion proteins in vitro and in vivo.

[0062] In an embodiment, the -TrCP chimeric further comprises a nucleic acid encoding a selfcleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the 0-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9.

[0063] In another aspect, this disclosure provides a retroviral CAR construct comprising a nucleic acid encoding an anti-tumor CAR, a nucleic acid encoding P-TrCP chimera, and a nucleic acid encoding a self-cleaving 2 A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera. In an embodiment, the nucleic acid encoding the self-cleaving 2A peptide is set forth in SEQ ID NO: 9. In some embodiments, the encoded P-TrCP chimera is a full-length P-TrCP protein encoded by the nucleic acid set forth in SEQ ID NO: 2. In an embodiment, the encoded P-TrCP chimera is a truncated P-TrCP protein lacking the WD40 domain (“P-TrCP (AWD)”), said P-TrCP chimera is encoded by the nucleic acid set forth in SEQ ID NO: 3. In some embodiments, the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and / or a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16. In an embodiment, the P-TrCP chimera is a full-length protein P-TrCP.10GS.SH2(C) or P-TrCP.10GS.SH2(N). In In some embodiments, the P-TrCP chimera is a truncated protein lacking the WD40 domain (“P-TrCP (AWD)”), wherein the P-TrCP chimera is P-TrCP(AWD).10GS.SH2(C), or P- TrCP(AWD).10GS.SH2(N). In an embodiment, the P-TrCP chimera is P-TrCP.10GS.SH2(C) or P- TrCP.10GS.SH2(N). In certain embodiments of the herein described retroviral CARs, the retroviral CAR constructs, further comprises a FKBP12F36domain (SEQ ID NO: 17) at the N-terminus of the nucleic acid encoding the P-TrCP chimera, wherein the nucleic acid encoding the P-TrCP chimera is set forth in SEQ ID NO: 2 or SEQ ID NO:3. In an embodiment of the herein described retroviral CARs, the CAR comprises a nucleic acid encoding an anti-tumor CAR is set forth in SEQ ID NO: 1P-661940-PC

[0064] The inventors have shown that targeted degradation of endogenous PD-1 by P-TrCP chimeras provided enhanced function to CAR-T cells. CAR-T cells expressing P-TrCP chimeras not only demonstrated reduced PD-1 on the cell surface, but also demonstrated increased proliferation even in the presence of PD-L1. The results show that P-TrCP chimeras effectively overcome PD-1 / PD-L1 suppression, and improve the efficacy of CAR-T cells in immunotherapy and for treating cancer.

[0065] The present disclosure discusses the regulatory roles of N-linked glycosylation in PD-1 protein stability, subcellular localization, and binding affinity with its ligand. The disclosure first shows a critical role of N-linked glycosylation in PD-1 protein stability, as disruption of glycosylation shortened PD-1 half-life. A total of four N-linked glycosylation sites (N49, N58, N74 and N116) are identified, and are demonstrated to cooperatively regulate for PD-1 stabilization. Glycosylation on these sites also dictates the intracellular localization of PD-1. Disruption of this modification on PD-1 alters their localization from membrane to exclusively cytoplasmic, and disables the immune checkpoint. N-linked glycosylation appeared to impede the interaction between PD-1 and PD-L1, suggesting that a dynamic cycle between glycosylation and deglycosylation of PD-1 may be a critical mechanism for PD-1 / PD-L1 signaling. The present disclosure shows protein kinase GSK3 and E3 ubiquitin ligase P-TrCP are important in regulating PD-1 stability. Of note, a single protein could be regulated by multiple kinases.

[0066] Ectopic targeting proteins for ubiquitination and subsequent proteasomal degradation is often used to interrogate the role of cellular proteins in normal and diseased state. Here, it was show that fusion of two SH2 domains from the SHP-2 phosphatase to P-TrCP E3 ubiquitin ligase effectively degraded glycosylated PD-1, overcoming the N-linked glycosylation-mediated protection of PD-1. Degradation of endogenous PD-1 was found to improve CAR-T cell proliferation. Since P-TrCP chimeras direct substrate degradation through targeting peptide (TP)- mediated recognition and binding, the inventors use a targeting peptide with high affinity and specificity for PD-1. Compared with gene knockout approaches, engineered P-TrCP chimeras reduce but do not completely eliminate intended targets. Therefore, it would not elicit the same severity of consequences as those raised from gene knockout. The integration of PD-1 -targeting P-TrCP chimeras into CAR T vectors improves the conventional CAR T technology. This modification enhances the expansion of CAR T cells and neutralizes the PD-1 immune checkpoint,P-661940-PC all without requiring additional therapeutic interventions like administering anti-PD-1 medications such as nivolumab (Opdivo) or pembrolizumab (Keytruda) to individuals with cancer.

[0067] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0068] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLESMATERIALS AND METHODSPlasmids, antibodies and reagents

[0069] Expression plasmid for FLAG-tagged ©-TpXIIwas described previously (as described by F. Cong, J. Zhang, W. Pao, P. Zhou, H. Varmus, A protein knockdown strategy to study the function of beta-catenin in tumorigenesis. BMC Mol Biol 4, 10 (2003)). Expression plasmid for PD-1 was purchased from Sino Biological Inc. (Beijing, China). Expression plasmid for GSK30 was constructed by cloning GSK3 H cDNA into pCMV.Tag2B vector. Retroviral CAR vector 4H1128z was a kind gift from Dr. Renier Brentj ens (Memorial Sloan Kettering Cancer Center, NY, USA).

[0070] Antibodies used for immunoblotting and co-immunoprecipitation included mouse anti- Myc (4A6, Millipore Sigma, St. Louis, MO, USA), mouse anti-FLAG (M2, Millipore Sigma), goat anti-human PD-1 (R&D Systems, Minneapolis, MN, USA), mouse anti-human GAPDH (7B, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-a-tubulin (Proteintech Group Inc, Rosemont, IL, USA) and rabbit anti- NK-ATPase (EP1845Y, Abeam, Cambridge, MA, USA). Antibodies used in flow cytometry are listed in the flow cytometry section.

[0071] Lysosome inhibitor chloroquine, ready-made solution of proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide were purchased from Millipore Sigma (St. Louis, MO, USA). N-linked glycosylation inhibitor Tunicamycin was purchased from Cell SignalingP-661940-PCTechnology (Danvers, MA, USA). N-linked glycosidase PNGase F and O-linked glycosidase were brought from New England Biolabs Inc. (Ipswich, MA, USA)Cell culture and transfection

[0072] Human embryonic kidney cell line HEK293T (American Type Culture Collection, Manassas, VA, USA) was cultured in Gibco™ Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS). Human ovarian cell lines SK-OV-3 WT and SK-OV-3-PD-L1 (kindly provided by Dr. Renier Brentjens) and human T lymphoblast Jurkat cells (ATCC) were cultured in Gibco™ RPMI 1640 Medium with 10% (v / v) FBS. All cells were grown at 37 °C supplied with humidified atmosphere containing 5% CO2. Plasmid DNA was transfected into cells using X-tremeGENE™ HP DNA Transfection Reagent (Roche) according to manufacturer’s protocol.Site-directed mutagenesis

[0073] Expression plasmids for PD-1 NQ mutants were generated by QuikChange Multi Site- Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, US). Mutagenic primers were designed using an online program QuikChange Primer Design(hitp.’ / Zwww genomics.agilent corn / primerDesig.nProgram.jsp).Western blotting

[0074] After harvest, cells were lysed with RIPA-150 buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 0.5% sodium deoxycholate) supplemented with protease inhibitor cocktails (Roche, Basel, Switzerland). Protein concentration of cell lysates was determined by Bradford method (Bio-Rad, Hercules, CA, US). Protein samples were separated by SDS-PAGE, followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes and incubated with primary antibody overnight and secondary antibodies for 1 hour at room temperature. Immunoblots were visualized using the Odyssey ® CLx Imaging System (LI-COR, Lincoln, Nebraska USA).Co-immunoprecipitation

[0075] Co-immunoprecipitation (co-IP) was carried out as previously described (W. W. Gao et al., Suppression of gluconeogenic gene transcription by SIKl-induced ubiquitination and degradation of CRTC1. Biochim Biophys Acta Gene Regul Meeh 1861, 211-223 (2018)). Briefly, cell samples were harvested and lysed with RIPA-150 buffer supplemented with protease inhibitor cocktailsP-661940-PC(Roche). Antibodies were incubated with cell lysate at 4 °C overnight, then recovered by incubating with recombinant protein G agarose (Thermo Fisher Scientific) for 3 hours at 4 °C. The protein G agarose was collected and washed three times with RIPA buffer. The immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting.Generation of Gammaretroviral Vectors

[0076] Engineered ®-TpXII chimeras were cloned into SFG-retroviral vector encoding the MUC16 ecto-targeted CAR, termed 4H1128z, to generate SFG-4H1128z / ®-TpXITvectors. Retroviral producer cell lines were generated with CaPO4 (Promega) to transiently transfect H29 packaging cells with forementioned retroviral constructs. Supernatant from the H29 cells was used to transduce 293Glv9 cells to generate stable retroviral producer cells lines.Generation of CAR Modified T Cells

[0077] Human T cells were cultured in RMPI-1640 medium supplemented with 10% (v / v) FBS (Atlanta Biologicals), 2 mM L-glutamine, 100 lU / mL penicillin, 100 pg / mL streptomycin (Invitrogen), and 100 lU / mL IL-2 (Proluekin, Novartis, Basel, Switzerland). Leukopacks from healthy donors were obtained from the New York Blood Center and buffy coats were isolated by density gradient centrifugation. T Cells were isolated from buffy coats using an EasySep Human T Cell Isolation Kit, resuspended at 1 x 106cells per mL, and activated with CTS Dynabeads at a 1 :2 cell to bead ratio. Dynabeads were magnetically removed after 48 hours and activated T cells were retrovirally transduced on RetroNectin (Takara) coated plates by spinnoculation over two consecutive days. CAR transduction was evaluated 7-10 days post activation by flow cytometry using an anti-idiotype antibody. CAR-T cells were fed by a complete media change every two days. All experiments were performed in compliance with all relevant ethical regulations and in accordance with IRB 95094.Flow Cytometry

[0078] Cells for flow cytometric analysis were resuspended in a total volume of 200 pL and stained with the following antibodies for 30 min at 4°C in the dark: mouse anti -human CD45 (eBiosciences clone 2D1, PE, 1:400), mouse anti-human CD69 (eBiosciences Clone FN50, FITC, 1 :400), mouse anti-human CD25 (eBiosciences clone BC96, PE-eFlour610, 1 :400), mouse anti-human CD279 (eBiosciences clone JI 06, Pe-Cyanine7, 1 :100), mouse anti -human CD4 (Biolegend clone RPA- T4, APC-Cyanine7, 1 :800), mouse anti-human CD8 (Invitrogen clone RPA-T8, PE-Cyanine5.5, 1 :800), Armenian hamster anti-4Hll (MSKCC Antibody Core Facility clone 22G6, Alexa647).P-661940-PCCells were washed once, resuspended in 1 ug / mL DAPT solution, and acquired on a 14 color, 16 parameter Attune Acoustic Focusing Flow Cytometer (Invitrogen) equipped with an autosampler.CAR-T Cell Expansion Assay

[0079] 1 x io5of SK-OV-3-PD-L1 cells were plated in a 12-well tissue culture treated plate and allowed to adhere overnight. 2 x 105CAR-positive T cells were then added and total media was brought to 4 mL without exogenous IL-2. After 2 days, half of the culture media was refreshed without exogenous IL-2. At the indicated timepoints, cells were harvested for enumeration and phenotyping by flow cytometry. 2 x 105CAR positive cells were measured out for restimulation on freshly plated SK-OV-3-PDL1 cells.Example 1. N-linked glycosylation stabilizes PD-1 protein by preventing GSK3p-induced degradation

[0080] In analysis of PD-1 protein sequence, it was noted that there is a consensus GSK3P (Glycogen synthase kinase 30) phosphorylation motif - SX(n)TX(n)S (S is serine, T is threonine and X is any amino acid) (E. Beurel, S. F. Grieco, R. S. Jope, Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacol Ther 148, 114-131 (2015)) in the extracellular domain (ECD) of PD-1 (Fig. 1A). As a key component in Wnt signaling pathway, GSK3P plays critical roles in embryonic development and tumorigenesis (B. W. Doble, J. R. Woodgett, GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116, 1175-1186 (2003)) through direct phosphorylation of downstream substrates such as b-catenin (S. Ikeda et al., Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3 beta-dependent phosphorylation ofbeta-catenin. EMBO 1 17, 1371-1384 (1998)), cyclin DI (B. P. Zhou et al., Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 6, 931-940 (2004)) and c-myc (M. A. Gregory, Y. Qi, S. R. Hann, Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization. J Biol Chem 278, 51606-51612 (2003)), and subsequent P-TrCP-mediated ubiquitination and degradation (C. Liu et al., beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation. Proc Natl Acad Sci U S A 96, 6273-6278 (1999), B. T. MacDonald, K. Tamai, X. He, Wnt / beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17, 9-26 (2009)), J. A. McCubrey et al., Multifaceted roles of GSK-3 and Wnt / beta-catenin in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention. Leukemia 28, 15-33 (2014)). The presence of GSK3P motif in PD-1 sequenceP-661940-PC suggested that PD-1 might be a potential substrate for GSK3p. To investigate whether PD-1 is regulated by GSK3P, increased amount of GSK3P was co-expressed with PD-1. Interestingly, although the major band of PD-1 was almost unaffected by GSK3P (Fig. IB, black dot), a faint lower band around 34 kDa dramatically decreased as the amount of GSK3P escalated (Fig. IB, black arrow). Such difference in the presence of GSK3P suggested the existence of at least two forms of PD-1 that respond differently to GSK3P-induced degradation.

[0081] The heterogeneous expression pattern of PD-1 protein usually suggests the presence of certain posttranslational modifications, most likely ubiquitination or glycosylation. To investigate whether PD-1 is glycosylated and to determine the type of glycosylation, HEK293T cell lysate containing overexpressed PD-1 protein was treated with either N-glycosidase (peptide-N- glycosidase F; PNGase F) or O-glycosidase. After PNGase F treatment, the major PD-1 band around 55 kDa (denoted by a black circle) almost completely disappeared, while the 34 kDa minor band (denoted by a black arrow) accumulated. The heterogeneous pattern of PD-1 was not greatly altered after O-glycosidase treatment, indicating that N-linked glycosylation is the dominant form on PD-1 protein (Fig. 1C). Furthermore, the addition of tunicamycin (TM), an N-linked glycosylation inhibitor, decreased the levels of the 55 kDa band and increased the levels of the 34 kDa band (Fig. ID). These results demonstrated that PD-1 is a highly glycosylated protein, with N-linked glycosylation being the major modification on it. The upper 55 kDa band corresponds to glycosylated form of PD-1, while the lower minor band at 34 kDa likely represents nonglycosylated PD-1.

[0082] To identify the putative N-glycosylation sites on PD-1, a search for NXT motifs, which are sites for N-linked glycosylation, was conducted. Four NXT motifs -N49, N58, N74, and N116 (Fig. IE)- are highly conserved across different species (Fig. 6). To confirm their roles in PD-1 glycosylation, the four N residues were mutated to glutamine (Q) individually or in combination. The four single mutants, N49Q, N58Q, N74Q, or N116Q, displayed moderate alterations in glycosylation pattern, as indicated by reduced size of glycosylated species when compared with wild-type (WT) PD-1 (as shown in Fig. IF, left panel). No detectable differences were found in the glycosylation pattern for three non-NXT NQ mutants (Fig. IF, right panel). Remarkably, PD- 1 glycosylation was almost completely ablated in PD-1 3NQ (N49Q / N58Q / N74Q) and 4NQ (N49Q / N58Q / N74Q / N116Q) mutants, as demonstrated by either significantly reduced heterogeneous pattern or dramatic shifted band (Fig. IF, left panel). These results indicate that allP-661940-PC four sites are involved in N-linked glycosylation on PD-1 and that they regulate this process cooperatively.

[0083] Given the increased levels of glycosylated PD-1 compared to its non-glycosylated form, investigated was whether glycosylation could stabilize PD-1 protein by introducing the protein synthesis inhibitor, cycloheximide (CHX), to control and tunicamycin-treated cells. As shown in Fig. 1G, the non-glycosylated PD-1 (denoted by black arrow) was rapidly turned over with a halflife of 2 hours while the glycosylated PD-1 (denoted by black circle) remained stable at the same period, indicating that glycosylated PD-1 proteins were more stable compared to their non- glycosylated counterparts (Fig. 1G, upper panel). The trend was also reflected in the turnover curve (Fig. 1G, lower panel), indicating that glycosylation could stabilize PD-1 protein. While this study was ongoing, Sun et al published the same finding on N-linked glycosylation-involved stabilization of PD-1 protein (L. Sun et al., Targeting Glycosylated PD-1 Induces Potent Antitumor Immunity. Cancer Res 80, 2298-2310 (2020)).

[0084] Since GSK3 cannot effectively degrade PD-1 WT (Fig. IB), there was an interest about its effect on glycosylation deficient PD-1 4NQ mutant. To this end, increasing amount of GSK30 was co-expressed with PD-1 4NQ mutant. In contrast to the slight effect on PD-1 WT (Fig. IB), PD-1 4NQ was dramatically destabilized by GSK3P (Fig. 1H). This difference between PD-1 WT and 4NQ indicates that glycosylation protects the PD-1 protein from GSK3p-induced degradation. Subsequent Co-IP experiment showed that glycosylation deficient PD-1 4NQ had a much higher binding affinity to GSK3P compared to PD-1 WT (Fig. 4D), suggesting that glycosylation inhibited the binding between PD-1 and GSK3p. Taken together, these results demonstrated that glycosylation stabilized PD-1 protein and prevented GSK3P-induced degradation by inhibiting their interaction.Example 2. N-linked glycosylated PD-1 is predominantly membrane localized

[0085] To determine whether glycosylation affects the intracellular localization of PD-1, the presence of PD-1 WT and NQ mutants in both the membrane and cytoplasmic compartments was investigated using a fractionation assay. It was found that PD-1 glycosylation-deficient mutants displayed more predominantly cytosolic accumulation than PD-1 WT protein, indicating reduced translocation from the cytoplasm to the cell surface after glycosylation blockade (Fig. 2A). Immunofluorescent staining also showed that PD-1 WT colocalized with the membrane marker NK-ATPase (Fig. 2B), while the N49Q mutant displayed increased cytoplasmic distribution (Fig.P-661940-PC2B) Significantly, when all four glycosylation sites were simultaneously disrupted, the resulting 4NQ mutant was exclusively localized in the cytoplasm. These findings suggest that glycosylation plays a crucial role in determining the intracellular localization of PD-1.Example 3. N-linked glycosylation interferes with PD-1 and PD-L1 interaction

[0086] N-linked glycosylation is known to play a crucial role in receptor-ligand interactions (J. C. Cheung, R. A. Reithmeier, Scanning N-glycosylation mutagenesis of membrane proteins. Methods 41, 451-459 (2007)). To investigate whether glycosylation modulates the binding between PD-1 and PD-L1, co-immunoprecipitation (co-IP) experiments were conducted. Both PD-1 WT and the glycosylation-deficient NQ mutants were detected in the PD-L1 immunoprecipitates (Fig. 3A). Remarkably, while the glycosylation-deficient PD-1 mutants were expressed much lower than those of glycosylated PD-1, the mutant forms were present at dramatically higher level in the PD- 1-PD-L1 complex (Fig. 3A), suggesting that glycosylation could inhibit the interaction between PD-1 and PD-L1. This finding was further supported by the result of TM treatment, where the inhibition of N-linked glycosylation significantly increased the binding between PD-1 and PD-L1 (Fig. 3B). Taken together, these results indicate that glycosylation plays a regulatory role in the PD-1-PD-L1 interaction.Example 4. Engineered P-TrCP chimera targets glycosylated PD-1 for degradationPD-1 signaling is known to suppress T cell proliferation, cytokine production and cytolytic activity, leading to T cell exhaustion (G. J. Freeman et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192, 1027-1034 (2000)). Consequently, several studies have targeted this signaling to improve CAR-T cell efficacy. CRISPR / Cas9-mediated PD-1 depletion has been found to enhance the anti-tumor efficacy of CAR T-cells (L. J. Rupp et al ., CRISPR / Cas9-mediated PD-1 disruption enhances anti -tumor efficacy of human chimeric antigen receptor T cells. Sci Rep 7, 737 (2017)). However, despite the improved efficacy, a concern about increased autoimmune response also arises due to the lack of “brake” effect from PD-1 signaling with this methodology (E. McGowan et al., PD-1 disrupted CAR-T cells in the treatment of solid tumors: Promises and challenges. Biomed Pharmacother 121, 109625 (2020)). Here, Protein Knockout Technology (PKO), the pioneered targeted protein degradation (TPD) strategy was employed to degrade endogenous PD- 1 (as described by P. Zhou, R. Bogacki, L. McReynolds, P. M. Howley, Harnessing theP-661940-PC ubiquitination machinery to target the degradation of specific cellular proteins. Mol Cell 6, 751 - 756 (2000); J. Zhang, N. Zheng, P. Zhou, Exploring the functional complexity of cellular proteins by protein knockout. Proc Natl Acad Sci U S A 100, 14127-14132 (2003)). SCFb-TrCP(Skpl- Cullinl-F-box) E3 ubiquitin ligases complex is known to mediate GSK3P phosphorylation- induced protein degradation such as P-catenin in Wnt signaling pathway (B. T. MacDonald, K. Tamai, X. He, Wnt / beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17, 9-26 (2009); J. A. McCubrey et al., Multifaceted roles of GSK-3 and Wnt / beta-catenin in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention. Leukemia 28, 15- 33 (2014). Among this complex, F-box protein P-TrCP is responsible for substrate recognition and binding through its WD40-repeat domain (C. Liu et al., beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation. Proc Natl Acad Sci U S A 96, 6273-6278 (1999)). By directly fusing P-TrCP protein with a targeting peptide (TP), a small fragment of peptide capable of binding to the substrate of interest, the resultant chimeric P-TrCP proteins could specifically recognize the intended substrates and target them for proteasome- mediated degradation (Fig. 4A).

[0087] Identifying an effective targeting peptide to the intended substrate is crucial for this TPD technology. Tyrosine phosphatase SHP-2 was reported as the major mediator for inhibitory activity of PD-1 (T. Yokosuka et al., Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J Exp Med 209, 1201-1217 (2012)). Following recruitment and binding to the cytoplasmic ITIM and ITSM motif of PD-1 (Fig. 4B, a), activated SHP-2 dephosphorylates downstream molecules in TCR and CD28 signaling pathways, such as PI3K and Ras, to transmit inhibitory signals (E. Hui et al., T cell costimulatory receptor CD28 is a primary target for PD-l-mediated inhibition. Science 355, 1428- 1433 (2017)). Two SH2 domains at N-terminal of SHP-2, SH2 (N) and SH2 (C), are required for PD-1 binding (Fig 4B, b) (N. Patsoukis et al., Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation. Commun Biol 3, 128 (2020)).

[0088] Both SH2 (N) and SH2 (C) domain of SHP-2 phosphatase were chosen as candidate targeting peptides. To generate chimeric 0-TrCP E3 ubiquitin ligase, three -TrCP expression vectors, 0-TrCPWT, P-TrCPWT.10GS and P-TrCP (AWD).IOGS were used (Fig. 4C). 10GS (SEQ ID NO: 10) is a small flexible fragment consisting of ten tandem repeats of glycine and serine, while P-TrCP(AWD) is a truncated mutant without the WD domain (Fig. 4C). Since F-boxP-661940-PC proteins bind to their substrates through the WD40-repeat domain (E. McGowan et al., PD-1 disrupted CAR-T cells in the treatment of solid tumors: Promises and challenges. Biomed Pharmacother 121, 109625 (2020)), this truncated mutant is unable to recognize and degrade endogenous P-TrCP substrates, thus targeting only the intended neo-substrate. By fusing the three -TrCP constructs with SH2 (N), SH2 (C) or SH2 (N) plus SH2 (C), a total of nine P-TrCP chimeras were generated. As shown in Fig. 4D, all chimeras effectively degraded glycosylated PD-1 proteins when compared with P-TrCP vector (Fig. 4D). Particularly, the SH2 (C)-containing chimeras P-TrCP.10GS.SH2 (C) and P-TrCP(AWD).10GS.SH2 (C) displayed higher activities for PD-1 degradation, suggesting that SH2 (C) domain may possess higher binding affinity to PD-1 than SH2 (N). In subsequent dose-dependent degradation assay, PD-1 protein levels decreased as the amount of P-TrCP.10GS.SH2 (C) (Fig. 4E, left panel) or P-TrCP(AWD). 10GS.SH2 (C) (Fig. 4E, right panel) increased. Both chimeras bound to PD-1 proteins in co-IP experiments (Fig. 4F). Taken together, by fusing P-TrCPE3 ubiquitin ligase with SHP-2 SH2 (C) domain as targeting peptide, the resulting chimeras could effectively induce the degradation of glycosylated PD-1 protein, overcoming the protection from N-linked glycosylation.Example 5. P-TrCP chimera promotes ex vivo expansion of CAR-T cells

[0089] It was posited that PD-1 targeting PKO could enhance the efficacy of CAR T-cells. To determine if P-TrCP chimeras could degrade endogenous PD-1 in T-cells, they were cloned into the pBMN.GFP retroviral vector and transduced into Jurkat cells. All 0-TrCP chimeras effectively degraded endogenous PD-1, especially for those with the 10GS fragment (Fig. 5A). To assess the effect of P-TrCP chimeras on CAR-T cells, P-TrCP.10GS.SH2 (C) and P-TrCP (AWD).10GS.SH2 (C) were cloned into the second-generation retroviral CAR vector. This CAR, labeled as 4H1128z, consists of a binding domain that recognizes ectodomain of MUC16 (MUC16ecto) and human CD28 plus CD3 zeta T-cell signaling domain (S. Rafiq et al., Targeted delivery of a PD-1 -blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36, 847-856 (2018)) (Fig. 5B, upper panel). A self-cleaving 2A peptide derived from porcine teschovirus, i.e. P2A (J. H. Kim et al., High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, el8556 (2011)), was used here to link CAR construct and P-TrCP chimera to allow the simultaneous expression of upstream CAR andP-661940-PC downstream 0-TrCP chimera (Fig. 5B, lower panel). Modified CAR constructs were named as 4H1128z / P-TrCP.10GS.SH2 (C) or 4H1128z / 0-TrCP(AWD).10GS.SH2 (C) respectively.

[0090] The engineered vectors were then transduced into primary human T-cells from three different donors. Following a period of rest, CAR-T cells expressing the indicated constructs were stimulated and PD-1 expression was measured. Both 4H1128z / P-TrCP.10GS.SH2 (C) and 4H1128z / p-TrCP(AWD).10GS.SH2 (C) could effectively degrade endogenous PD-1 when compared with unmodified 4H1128z CAR-T cells (Fig. 5C). However, a poor expression of 4H1128z / p-TrCP.10GS.SH2 (C) was found possibly due to its large size. Hence, only 4H1128z / 0- TrCP(AWD).10GS.SH2 (C) was used in further studies and designated as CAR-PKO in following figures. Flow cytometry analysis demonstrated that P-TrCP(AWD). 10GS.SH2 (C) chimera did not affect CAR expression on the cell surface (Fig. 5D). More importantly, the percentage of PD- 1 positive CAR T-cells in P-TrCP chimera-expressing group dramatically decreased compared to control empty CAR vector group (Fig. 5E). Even for PD-1 positive CAR T-cells in two groups, the expression intensity of PD-1 in 0-TrCP chimera-expressing group was still significantly lower than that in control empty CAR vector group (Fig. 5F). Taken together, both Western blot and flow cytometry results demonstrated that our 0-TrCP chimera effectively decreased endogenous PD-1 levels in CAR T-cells.

[0091] Next, to investigate whether targeted degradation of endogenous PD-1 by 0-TrCP chimeras conferred any enhanced function to CAR-T cells, SK-OV-3 cells stably expressing PD-L1 (SK- OV-3-PD-L1) were co-cultured with 0-TrCP chimera-modified CAR-T cells. CAR-T cells were harvested at the indicated time points following coculture for enumeration and phenotyping by flow cytometry analysis. Compared to unmodified 4H1128z CAR-T cells, those expressing 0- TrCP chimeras not only demonstrated reduced PD-1 on the cell surface (Fig. 5G) but also, importantly, showed increased ex vivo proliferation even in the presence of PD-L1 (Fig. 5H), suggesting that 0-TrCP chimeras could effectively overcome PD-1 / PD-L1 signaling-mediated proliferative suppression, which would improve T-cell exhaustion caused by this signaling and enhance efficacy of CAR-T cells in immunosuppressive TME.DISCUSSION OF EXAMPLES

[0092] In the present examples, the inventors explored the regulatory roles of N-linked glycosylation on PD-1 protein stability, subcellular localization, and binding affinity with its ligandP-661940-PCPD-L1. It was demonstrated that N-linked glycosylation is crucial for the stability of PD-1 ; disrupting glycosylation significantly reduced the half-life of PD-1. Four N-linked glycosylation sites (N49, N58, N74, and N116) that cooperatively contribute to PD-1 stabilization were identified. Additionally, glycosylation at these sites influenced the intracellular localization of PD- 1. Disruption of this modification resulted in a shift of PD-1 localization from the membrane to exclusively cytoplasmic.

[0093] Our co-immunoprecipitation (Co-IP) experiment indicated that N-linked glycosylation may inhibit the interaction between PD-1 and PD-L1, suggesting that a dynamic cycle of glycosylation and deglycosylation could be a potential mechanism for PD-1 / PD-L1 signaling. Cellular components regulating this cycle might be potential targets for PD-1 signaling-directed therapy. Further investigations revealed that protein kinase GSK3P and E3 ubiquitin ligase P-TrCP play central roles in regulating PD-1 stability. Notably, a single downstream substrate could be influenced by multiple upstream kinases. It remains unclear whether other kinases are also involved in PD-1 regulation, particularly since GSK3P-mediated phosphorylation requires prior phosphorylation by other kinases, such as CK1 (C. Liu et al., Control of beta-catenin phosphorylation / degradation by a dual-kinase mechanism. Cell 108, 837-847 (2002)).

[0094] An alternative targeted protein degradation (TPD) strategy known as protein knockout (PKO) was introduced. In this approach, a targeting peptide (TP), consisting of several to hundreds of amino acids linked to E3 ubiquitin ligase by a linker, is used to recruit and bind the target substrate, facilitating its proteasomal degradation. Unlike PROTAC, PKO does not require specific pockets or grooves on target proteins for small molecule docking; it only needs to ensure the binding between the targeting peptide and the substrate. Moreover, unlike RNA interference (RNAi), which reduces only newly synthesized proteins, PKO targets both newly synthesized and existing proteins. This is crucial for long-lived targets, as they can remain functional for some time even after the introduction of siRNA knockdown strategies. This advantage makes PKO a more effective strategy for the rapid degradation of target proteins and for providing rapid experimental readouts.

[0095] In the examples, it was demonstrated that the fusion of two SH2 domains from SHP-2 phosphatase with the P-TrCP E3 ubiquitin ligase as a targeting peptide effectively degraded glycosylated PD-1. As the SH2 domains target the cytosolic ITIM and ITSM domains of PD-1, the 0-TrCP chimera construct was able to overcome the ectocytic N-linked glycosylation-mediatedP-661940-PC protection on the PD-1 protein. ThePKO strategy relies on substrate degradation through targeting peptide (TP)-mediated recognition and binding, thus, identifying a targeting peptide with high sensitivity and specificity for the substrate presents a major challenge for this approach. Additionally, the size of the targeting peptide is another critical consideration for this technique; the SH2 (C) domain used here is approximately 300 amino acids long. Further investigations are needed to determine if there are smaller targeting peptide candidates with higher sensitivity and specificity for PD-1.

[0096] Compared with the gene knockout approach, engineered 0-TrCP chimeras reduce but do not completely eliminate intended targets. Therefore, they are unlikely to elicit the same severity of consequences as those arising from gene knockout. For example, while complete depletion of endogenous PD-1 in CAR T-cells via CRISPR knockout significantly enhanced their efficacy, it also raised new concerns about an enhanced autoimmune response due to the absence of the "brake" effect from PD-1 signaling (as described by N. Patsoukis, et al. Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation. Commun Biol. 2020 Mar 17;3(1):128. doi:10.1038 / s42003-020-0845-0). Overall, our findings not only further emphasize the regulatory roles of N-linked glycosylation in PD-1 protein functions but also, more importantly, suggest that targeting PD-1 for degradation through the PKO strategy could be a viable therapeutic option in CAR T-cell-mediated cancer immunotherapy.EXAMPLE 6Materials & MethodsPlasmids, antibodies and reagents

[0097] Expression plasmid for FLAG-tagged 0-TrCP was described previously (as described by Feng Cong, et al.. A protein knockdown strategy to study the function of beta-catenin in tumorigenesis. BMC Mol Biol. 2003 Sep 29; 4: 10. doi: 10.1186 / 1471-2199-4-10, which is incorporated herein by reference in its entirety). Expression plasmid for PD-1 was purchased from Sino Biological Inc. (Beijing, China). Expression plasmid for GSK30 was constructed by cloning GSK30 cDNA into pCMV.Tag2B vector. Retroviral CAR vector 4H1128z was a kind gift from Dr. Renier Brentjens (Memorial Sloan Kettering Cancer Center, NY, USA).

[0098] Antibodies used for immunoblotting and co-immunoprecipitation included mouse anti- Myc (4A6, Millipore Sigma, St. Louis, MO, USA), mouse anti-FLAG (M2, Millipore Sigma), goatP-661940-PC anti-human PD-1 (R&D Systems, Minneapolis, MN, USA), mouse anti-human GAPDH (7B, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-oc-tubulin (Proteintech Group Inc, Rosemont, IL, USA) and rabbit anti-NK-ATPase (EP1845Y, Abeam, Cambridge, MA, USA). Antibodies used in flow cytometry are listed in the flow cytometry section.

[0099] Lysosome inhibitor chloroquine, ready-made solution of proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide were purchased from Millipore Sigma (St. Louis, MO, USA). N-linked glycosylation inhibitor Tunicamycin was purchased from Cell Signaling Technology (Danvers, MA, USA). N-linked glycosidase PNGase F and O-linked glycosidase were bought from New England Biolabs Inc. (Ipswich, MA, USA). ImmunoCult™ Human CD3 / CD28 T Cell Activator and EasySep Human T Cell Isolation Kit was bought from STEMCELL TECHNOLOGIES (Vancouver, British Columbia, Canada). Lonza SE Cell Line 4D Nucleofector™ X Kit was purchased from Lonza (Basel, Switzerland).Cell culture and transfection[000100] Human embryonic kidney cell line HEK293T (American Type Culture Collection, Manassas, VA, USA) was cultured in Gibco™ Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS). Human ovarian cell lines SK-OV-3 WT and SK-OV-3-PD-L1 (kindly provided by Dr. Renier Brentj ens) and human T lymphoblast lurkat cells (ATCC) were cultured in Gibco™ RPMI 1640 Medium withl0% (v / v) FBS. All cells were grown at 37°C supplied with humidified atmosphere containing 5% CO2. Plasmid DNA was transfected into cells using X-tremeGENE™ HP DNA Transfection Reagent (Roche) according to manufacturer’s protocol.Electroporation[000101] Electroporation was performed with Lonza SE Cell Line 4D-Nucleofector™ X Kit on Lonza 4D-Nucleofector as per manufacture’s guidance. Specifically, 1 x 106 / ml Jurkat T lymphocytes were activated by ImmunoCult™ Human CD3 / CD28 T Cell Activator for 3 days. After that, totally 1 x 106 cells were harvested and centrifuged at 90 g for 10 mins. The pellet was resuspended with 2 pg plasmid DNA and 100 pl SE Cell Line Solution. Program CL-120 was used for nucleofection. After 48 hours incubation, Jurkat cells were harvested and lysed for Western blot analysis.P-661940-PCSite-directed mutagenesis[000102] Expression plasmids for PD-1 NQ mutants were generated by QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, US). Mutagenic primers were designed using an online program QuikChange Primer Design(hitp: / / www. genomics. agiknt.com / primerDesignProgram.jsp).Western blotting[000103] After harvest, cells were lysed with SDS lysis buffer (50 mM Tris (pH 8.1), 1% SDS, 5 mM EDTA) supplemented with protease inhibitor cocktails (Roche, Basel, Switzerland). Protein concentration of cell lysates was determined by Bradford method (Bio-Rad, Hercules, CA, US). Protein samples were separated by SDS-PAGE, followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes and incubated with primary antibody overnight and secondary antibodies for 1 hour at room temperature. Immunoblots were visualized using the Odyssey ® CLx Imaging System (LI-COR, Lincoln, Nebraska USA).Co-immunoprecipitation[000104] Co-immunoprecipitation (co-IP) was carried out as previously described by Weiwei Gao, et al. Suppression of gluconeogenic gene transcription by SIKl-induced ubiquitination and degradation of CRTC1. Biochim Biophys Acta Gene Regul Meeh. 2018 Mar; 1861(3):211-223, which is incorporated herein by reference in its entirety. Briefly, cell samples were harvested and lysed with Western & IP buffer (20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor cocktails (Roche). 1 pg antibodies or Fc- fusion proteins were incubated with cell lysate at 4 °C overnight, then recovered by incubating with recombinant protein G agarose (Thermo Fisher Scientific) for 3 hours at 4 °C. The protein G agarose was collected and washed three times with Western & IP buffer. The immunoprecipitates were separated by SDS-PAGE and analyzed by western blotting.Generation of Gammaretroviral Vectors[000105] Engineered 0-TrCP chimeras were cloned into SFG-retroviral vector encoding the MUC16 ecto-targeted CAR, termed 4H1128z, to generate SFG-4H1128z / p-TrCP vectors. Retroviral producer cell lines were generated with CaPO4 (Promega) to transiently transfect H29P-661940-PC packaging cells with forementioned retroviral constructs. Supernatant from the H29 cells was used to transduce 293Glv9 cells to generate stable retroviral producer cells lines.Generation of CAR Modified T Cells[000106] Human T cells were cultured in RMPI-1640 medium supplemented with 10% (v / v) FBS (Atlanta Biologicals), 2 mM L-glutamine, 100 lU / mL penicillin, 100 pg / mL streptomycin (Invitrogen), and 100 JU / mL IL-2 (Proluekin, Novartis, Basel, Switzerland). Leukopacks from healthy donors were obtained from the New York Blood Center and buffy coats were isolated by density gradient centrifugation. T Cells were isolated from buffy coats using an EasySep Human T Cell Isolation Kit, resuspended at 1 x 106cells per mL, and activated with CTS Dynabeads at a 1 :2 cell to bead ratio. Dynabeads were magnetically removed after 48 hours and activated T cells were retrovirally transduced on RetroNectin (Takara) coated plates by spinnoculation over two consecutive days. CAR transduction was evaluated 7-10 days post activation by flow cytometry using an anti-idiotype antibody. CAR-T cells were fed by a complete media change every two days. All experiments were performed in compliance with all relevant ethical regulations and in accordance with IRB 95094.Flow Cytometry[000107] Cells for flow cytometric analysis were resuspended in a total volume of 200 pL and stained with the following antibodies for 30 min at 4°C in the dark: mouse anti-human CD45 (eBiosciences clone 2D1, PE, 1:400), mouse anti-human CD69 (eBiosciences Clone FN50, FITC, 1 :400), mouse anti-human CD25 (eBiosciences clone BC96, PE-eFlour610, 1 :400), mouse antihuman CD279 (eBiosciences clone J106, Pe-Cyanine7, 1 :100), mouse anti-human CD4 (Biolegend clone RPA-T4, APC-Cyanine7, 1 :800), mouse anti-human CD8 (Invitrogen clone RPA-T8, PE-Cyanine5.5, 1 :800), Armenian hamster anti-4Hl 1 (MSKCC Antibody Core Facility clone 22G6, Alexa647). Cells were washed once, resuspended in 1 ug / mL DAPI solution, and acquired on a 14 color, 16 parameter Attune Acoustic Focusing Flow Cytometer (Invitrogen) equipped with an autosampler.CAR-T Cell Expansion Assay[000108] 1 x 105 of SK-OV-3-PD-L1 cells were plated in a 12-well tissue culture treated plate and allowed to adhere overnight. 2 x 105 CAR-positive T cells were then added and totalP-661940-PC media was brought to 4 mL without exogenous TL-2. After 2 days, half of the culture media was refreshed without exogenous IL-2. At the indicated timepoints, cells were harvested for enumeration and phenotyping by flow cytometry. 2 * 105 CAR positive cells were measured out for restimulation on freshly plated SK-OV-3-PDL1 cells.ResultsN-linked glycosylation stabilizes PD-1 by preventing GSK30-mediated degradation[000109] To investigate the regulatory mechanisms that control PD-1 threshold levels, its post-translational modifications were examined and a putative GSK30 (glycogen synthase kinase 30) phosphorylation motif — S / T-X(n)-S / T-X(n)-S / T (where S represents serine, T threonine, and X any amino acid) (as described by Chia-Wei Li, et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat Commun. 2016 Aug 30:7: 12632. doi: 10.1038 / ncommsl2632, which is incorporated herein by reference in its entirety) — within the N- terminal region (amino acids 113-133) of PD-1 was identified. (Fig. 8A). As a key component in Wnt signaling pathway, GSK3P plays critical roles in embryonic development and tumorigenesis through direct phosphorylation of downstream substrates such as P-catenin, cyclin DI and c-myc (as described by Bradley W Doble, et al. GSK-3: Tricks of the Trade for a Multi-Tasking Kinase. J Cell Sci. 2003 Apr l;116(Pt 7):1175-86. doi: 10.1242 / jcs.00384; Binhua P Zhou, et al. Dual Regulation of Snail by GSK-3beta-mediated Phosphorylation in Control of Epithelial- Mesenchymal Transition. Nat Cell Biol. 2004 Oct;6(10):931-40. doi: 10.1038 / ncbll73; and Qingqing Ding, et al. Degradation of Mcl-1 by beta-TrCP Mediates Glycogen Synthase Kinase 3- induced Tumor Suppression and Chemosensitization. Mol Cell Biol. 2007 Jun;27(ll):4006-17. doi: 10.1128 / MCB.00620-06, each of which is incorporated herein by reference in its entirety). The presence of GSK3P phosphorylation motif in PD-1 sequence suggested that PD-1 might be a potential substrate for GSK3p. To investigate whether PD-1 is regulated by GSK3P, increasing amount of GSK3P was coexpressed with PD-1. Interestingly, although the major PD-1 band was largely unaffected by GSK3P overexpression (Fig. 8B, black dot), a lower band at approximately 34 kDa — corresponding to the predicted molecular weight — was markedly reduced in a GSK3 dose-dependent manner (Fig. 8B, black arrow). The differential response to GSK3 suggested the presence of at least two distinct PD-1 species, prompting further investigation into the identity of the 34 kDa species and its heightened sensitivity to GSK30-mediated degradation.P-661940-PC[000110] The heterogeneous expression pattern of PD-1 protein usually suggests the presence of certain posttranslational modifications, most likely ubiquitination or glycosylation (as described by Chia-Wei Li, et al. supra). To investigate whether PD-1 is glycosylated and to determine the type of glycosylation, HEK293T cell lysate containing overexpressed PD-1 protein was treated with either N-glycosidase (peptide-N-glycosidase F; PNGase F) or O-glycosidase. Following PNGase F treatment, the major PD-1 band at ~55 kDa (denoted by a black circle) was almost completely lost, while the 34 kDa minor band (denoted by a black arrow) accumulated. The heterogeneous pattern of PD-1 was not greatly altered after O-glycosidase treatment, indicating that N-linked glycosylation is the dominant form on PD-1 protein (Fig. 9A). Furthermore, the addition of tunicamycin (TM), an N-linked glycosylation inhibitor, decreased the levels of the 55 kDa band and increased the levels of the 34 kDa band (Fig. 9B). These results demonstrate that PD-1 is a highly glycosylated protein, with N-linked glycosylation as its predominant modification. The upper 55 kDa band corresponds to glycosylated form of PD-1, while the lower minor band at 34 kDa likely represents non-glycosylated PD-1.[000111] To identify N-glycosylation sites on PD-1, a search for NXT motifs was conducted and found four candidates: N49, N58, N74, and N116 (Fig. 9C), with N49, N74, and N116 conserved across species (Fig. 9D). Mutation of each site to glutamine (Q) moderately reduced glycosylation, while the 4NQ mutant (N49Q / N58Q / N74Q / N116Q) nearly eliminated it (Fig. 9E). Next assessed was whether glycosylation stabilizes PD-1 by treating cells with cycloheximide (CHX). Non-glycosylated PD-1 was rapidly degraded, while glycosylated PD-1 remained stable (Fig. 9F), suggesting that N-linked glycosylation protects PD-1 from turnover. While this study was underway, Sun et al. reported that glycosylation stabilizes PD-1 proteins, providing important insight into the post-translational regulation of this immune checkpoint receptor (as described by Sun et al., supra). The present findings, obtained independently and in parallel, are consistent with and further reinforce their observations.[000112] Given that GSK3P had minimal impact on the stability of glycosylated PD-1 (Fig. 8B), next examined was its effect on the glycosylation-deficient PD-1 4NQ mutant. Co-expression of increasing amounts of GSK3P with PD-1 4NQ resulted in marked protein destabilization, in stark contrast to the slight reduction observed with glycosylated PD-1 WT (Fig. 8C). A similar selective effect was observed for endogenous PD-1 in human Jurkat T cells, where GSK3P promoted degradation of the unglycosylated form (indicated by a black arrow), while sparing theP-661940-PC glycosylated form (indicated by a black circle) (Fig. 8D). These findings suggest that N-linked glycosylation protects PD-1 from GSK3p-mediated degradation. Supporting this notion, coimmunoprecipitation assays demonstrated that the PD-1 4NQ mutant exhibited markedly enhanced binding to GSK30 compared to wild-type PD-1 (Fig. 8E), suggesting that glycosylation impairs GSK3P association. Collectively, these data demonstrate that glycosylation stabilizes PD- 1 by inhibiting its association with GSK3p.[000113] To further investigate the correlation between GSK3P-mediated phosphorylation and PD-1 glycosylation, putative phosphorylation sites were mutated — SI 18, T120, and SI 27 — in predicted GSK3P motif by substituting serine / threonine residues with alanine (A), either individually or in combination (Figs. 8A and Fig. 8F). These phosphorylation-deficient mutants, along with PD-1 WT, were co-expressed in the presence or absence of GSK3p. As expected, glycosylated PD-1 WT exhibited modest degradation upon GSK3P co-expression, whereas all phosphorylation-deficient mutants remained largely unaffected, implicating the involvement of these sites in GSK3p-induced PD-1 destabilization (Fig. IF). In addition to enhanced stability, a pronounced band shift in the S118A / T120A mutant also was observed. However, given the intrinsic resistance of glycosylated PD-1 to GSK3P-mediated degradation, these mutations alone could not clearly delineate the functional importance of the phosphorylation sites.[000114] To better assess their contribution, the same S / T-to-A mutations were introduced into the PD-1 4NQ mutant, which is more susceptible to GSK30-induced destabilization (Fig. 8C). Notably, these combined mutations significantly increased the stability of the resulting phosphodeficient 4NQ mutant compared to PD-1 4NQ alone (Fig. 8G), further underscoring the role of SI 18, T120, and S127 in GSK3P-mediated degradation. Moreover, comparison of SI 18A / T120A mutant with various glycosylation-deficient mutants revealed that the mobility shift corresponded to a lower glycosylation state (Fig. 8H), suggesting that phosphorylation at these sites may be required for efficient PD-1 glycosylation.N-linked glycosylation modulates PD-1 subcellular localization and attenuates its interaction with PD-L1[000115] To assess whether N-linked glycosylation influences PD-1 subcellular localization, cellular fractionation assays were performed to compare the distribution of wild-type (WT) PD-1 and glycosylation-deficient NQ mutants. Glycosylation-deficient PD-1 proteins exhibitedP-661940-PC enhanced accumulation in the cytoplasmic fraction relative to WT, suggesting impaired trafficking from the cytosol to the plasma membrane upon loss of glycosylation (Fig. 11A). Consistently, immunofluorescence staining revealed that WT PD-1 colocalized with the membrane marker Na+ / K+-ATPase, whereas the N49Q mutant displayed increased cytoplasmic localization (Fig. 11B). Notably, the 4NQ mutant, in which all four glycosylation sites were disrupted, exhibited exclusive cytoplasmic distribution. Together, these results indicate that N-linked glycosylation is essential for proper subcellular trafficking and membrane localization of PD-1.[000116] Beyond its role in subcellular localization, N-linked glycosylation is known to critically influence receptor-ligand interactions (as described by Cheung., J.C., supra). To assess whether glycosylation modulates PD-1 binding to its ligand PD-L1, co-immunoprecipitation (coIP) assays were performed. Although glycosylation-deficient PD-1 mutants were expressed at lower levels in whole-cell lysates compared to wild-type PD-1, they were enriched to a much greater extent in PD-L1 immunoprecipitates (Fig. 10A), suggesting enhanced binding affinity in the absence of glycosylation. Consistent with this observation, treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, significantly increased PD-1 association with PD-L1 (Fig. 10B). To further quantify this interaction, pull-down assays were performed using recombinant PD-Ll-Fc fusion protein. Following normalization of PD-1 input and pull-down signals, relative binding efficiencies were calculated for all single and combinatorial N-to-Q mutants. While the N49Q andN58Q mutants showed similar binding to PD-Ll-Fc as WT PD-1, theN74Q and Ni l 6Q mutants exhibited markedly increased association, implicating these sites as key negative regulators of the PD-1-PD-L1 interaction (Fig. 13A-13B). Moreover, multi-site mutants (2NQ, 3NQ, and 4NQ) demonstrated progressively enhanced binding to PD-Ll-Fc compared to WT (Fig. 13A-13B). Collectively, these results indicate that N-linked glycosylation attenuates PD-1-PD-L1 binding, likely through steric or conformational hindrance at specific glycosylation sites.Targeting glycosylated, degradation-resistant PD-1 via engineered P-TrCP chimeric ligases[000117] PD-1 signaling suppresses T cell proliferation, cytokine production, and cytolytic activity, ultimately contributing to T cell exhaustion (as described by G. J. Freeman et al., supra). Consequently, several strategies were adopted to target this pathway to enhance the efficacy of chimeric antigen receptor (CAR)-T cells. For instance, CRISPR / Cas9-mediated deletion of PD-1 has been shown to improve the antitumor activity of CAR-T cells (K Abiko, et al. IFN-y fromP-661940-PC lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer. Br J Cancer. 2015 Apr 28; 112(9): 1501-9. doi: 10.1038 / bjc.2015.101). However, this approach raises concerns regarding potential autoimmune responses due to the loss of PD-l’s immunoregulatory “brake” function (as described by O. O. Yeku, et al. Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment. Sci Rep. 2017 Sep 5;7(1): 10541. doi: 10.1038 / s41598- 017-10940-8).[000118] Targeted protein degradation (TPD) is emerging as a powerful strategy to eliminate disease-relevant proteins. While Proteolysis Targeting Chimeras (PROTACs) are widely used, their reliance on ligandable pockets limits target scope. To address these limitations, Protein Knockout (PKO) technology, a pioneering TPD strategy, was introduced to degrade endogenous PD-1. The SCFp-TrCP (Skpl-Cullinl-F-box) E3 ubiquitin ligase complex mediates the degradation of GSK3P-phosphorylated substrates such as P-catenin in the Wnt signaling pathway (as described by Qingqing Ding, et al. Degradation of Mcl-1 by beta-TrCP Mediates Glycogen Synthase Kinase 3-induced Tumor Suppression and Chemosensitization. Mol Cell Biol. 471 2007 Jun;27(l l):4006-17. doi: 10. 1128 / MCB.00620-06 ; Patricia Rada, et al. SCF / {beta}-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keapl -independent manner. Mol Cell Biol. 2011 Mar;31(6):1121-33. doi: 10.1128 / MCB.01204- 10; and Bryan T MacDonald, et al. Signaling: Components, Mechanisms, and Diseases. Dev Cell. 2009 Jul;17(l):9-26. doi: 10.1016 / j.devcel.2009.06.016, each of which is incorporated herein by reference in its entirety). Within this complex, the F-box protein P-TrCP confers substrate specificity via its WD40 repeat domain. By fusing P-TrCP to a targeting peptide (TP) — a short peptide that binds the protein of interest — chimeric P-TrCP constructs capable of selectively recognizing and degrading PD-1 via the proteasome pathway (Fig. 12A) were generated.[000119] Identifying an effective targeting peptide to the intended substrate is crucial for this technology. Tyrosine phosphatase SHP-2 was reported as the major mediator for inhibitory activity of PD-1 (as described by Yokosuka, et al. Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J Exp Med (2012) 209 (6): 1201-1217. Following recruitment and binding to the cytoplasmic ITIM and ITSM motif of PD-1 (Fig. 12B), SHP-2 dephosphorylates downstream molecules in TCR and CD28 signaling pathways, such as PI3K and Ras, to transmit inhibitory signals (as described by sEnfu Hui, et al. T cell costimulatory receptor CD28 is a primary target for PD-1 -mediatedP-661940-PC inhibition. Science 2017 Mar 31 ;355(6332): 1428-1433. doi: 10.1126 / science.aafl 292, which is incorporated herein in its entirety). Two SH2 domains at N-terminal of SHP-2, SH2 (N) and SH2 (C), are required for PD-1 binding (Fig 12B) (as described by N. Patsoukis et al., Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation. Commun Biol 3, 128 (2020), which is incorporated herein by reference in its entirety).[000120] Both SH2 (N) and SH2 (C) domain of SHP-2 phosphatase were chosen as candidate targeting peptides. To generate chimeric 0-TrCP E3 ubiquitin ligase, three P-TrCP expression vectors, P-TrCP (no linker), P-TrCP.10GS and P-TrCP (AWD).10GS were used (Fig. 12C). 10GS is a small flexible fragment consisting of ten tandem repeats of glycine and serine, while P-TrCP (AWD) is a truncated mutant without the WD40 domain (Fig. 12C). Since P-TrCP binds its substrates via the WD40 domain, the truncated mutant lacking this domain cannot recognize or degrade endogenous P-TrCP substrates, thereby selectively targeting only the intended substrate (as described by J. Zhang, supra). By fusing the three P-TrCP constructs with SH2 (N), SH2 (C) or SH2 (N) plus SH2 (C), a total of nine P-TrCP chimeras were generated. As shown in Fig. 12D, all chimeras effectively degraded glycosylated PD-1 proteins when compared with basal vectors. Particularly, the SH2 (C)-containing chimeras P-TrCP.10GS.SH2 (C) and P-TrCP (AWD).10GS.SH2 (C) displayed higher activities for PD-1 degradation, suggesting that SH2 (C) domain may possess higher binding affinity to PD-1 than SH2 (N). In subsequent dose-dependent degradation assays, PD-1 protein levels decreased with increasing amounts of P-TrCP.10GS.SH2 (C) (Fig. 12E, upper panel) or P-TrCP(AWD).10GS.SH2 (C) (Fig. 12E, lower panel). In addition to highly glycosylated PD-1 WT protein, the glycosylation-deficient PD-1 4NQ mutant were also effectively degraded by P-TrCP.10GS.SH2 (C) chimera (Fig. 12F). Both chimeric constructs bound to PD-1 in co-immunoprecipitation (co-IP) assays (Fig. 12G). Taken together, fusion of the P-TrCP E3 ubiquitin ligase with the SHP-2 SH2(C) domain as a targeting peptide enabled the resulting chimeras to effectively induce degradation of glycosylated PD-1 by targeting their intracellular domain, thereby overcoming the protective effect conferred by N-linked glycosylation.P-TrCP chimera promotes ex vivo expansion of CAR-T cells[000121] It was hypothesized that PD-1 -targeting PKO could enhance the efficacy of CAR- T cells. To evaluate whether [3-TrCP chimeras can degrade endogenous PD-1 in T cells, the constructs were cloned into the pBMN.GFP retroviral vector and transduced into Jurkat cells. AllP-661940-PC0-TrCP chimeras effectively reduced endogenous PD-1 levels, with the greatest degradation observed in constructs containing the 10GS linker (Fig. 14A).[000122] For the application of PKO in CAR-T therapy, our goal is to design a single CAR-T vector capable of concurrently delivering both CAR and PKO, thereby obviating the need for separate therapeutic regimens involving CAR-T and anti-PD-1 therapies. To assess the functional impact of 0-TrCP chimeras in CAR-T cells, P-TrCP.10GS.SH2(C) and P- TrCP(AWD).10GS.SH2(C) were cloned into a second-generation retroviral CAR vector. This CAR, designated 4H1128z, consists of the extracellular 4H11 scFv, which targets the ectodomain of the MUC16 (MUC16ecto) tumor-associated antigen — commonly overexpressed in ovarian, pancreatic, and certain lung and breast cancers — and an intracellular signaling module comprising the human CD28 and CD3C domains, (as described by Alena A Chekmasova 1, Clin Cancer Res. 2010 Jul 15; 16(14):3594-606. doi: 10.1158 / 1078-0432.CCR-10-0192. Epub 2010 Jul 13 and Sarwish Rafiq, et al. Targeted delivery of a PD-l-blocking scFv by CAR-T cells enhances antitumor efficacy in vivo. Nat Biotechnol. 2018 Oct; 36(9): 847-504 856. doi: 10.1038 / nbt.4195, which is incorporated herein by reference in its entirety) (Fig. 14B, upper panel). A self-cleaving 2A peptide derived from porcine teschovirus (P2A) (as described by Jin Hee Kim, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus- 1 in human cell lines, zebrafish and mice. PLoS One. 201 l;6(4):el8556. doi: 10.1371 / journal. pone.0018556. Epub 2011 Apr 29, which is incorporated herein by reference in its entirety) was employed to link the CAR construct with the P-TrCP chimera, enabling simultaneous expression of the upstream CAR and the downstream P-TrCP chimera (Fig. 14B, lower panel). The modified constructs were designated 4H1128z / p-TrCP.10GS.SH2(C) and 4H1128z / p-TrCP(AWD).10GS.SH2(C), respectively.[000123] Engineered CAR constructs were transduced into primary human T cells derived from three independent donors. After a resting period, CAR-T cells expressing the indicated constructs were stimulated, and PD-1 expression was assessed. Both 4H1128z / p-TrCP.10GS.SH2(C) and 4H1128z / p-TrCP(AWD).10GS.SH2(C) efficiently degraded endogenous PD-1 compared with unmodified 4H1128z CAR-T cells (Figs. 14C(a)-14C(c)). However, poor expression of 4H1128z / p-TrCP.10GS.SH2(C) was observed, likely due to its large size. Therefore, only 4H1128z / p-TrCP(AWD).10GS.SH2(C) was used in subsequent experiments and is hereafter referred to as CAR-PKO.P-661940-PC[000124] Flow cytometry analysis confirmed that the expression of P-TrCP(AWD).10GS.SH2(C) did not alter the percentage of CAR-positive cells. However, reduced CAR expression was observed, likely due to the larger cargo size of the CAR-T retroviral vector (Fig. 4D). Importantly, the proportion of PD-1+ CAR T cells was markedly reduced in the CAR-PKO group compared with the empty vector control (Fig. 14E). Furthermore, among PD-1+ CAR-T cells, the mean fluorescence intensity of PD-1 was significantly lower in the P-TrCP chimera-expressing group (Fig. 14F). Collectively, both Western blot and flow cytometry data demonstrated that the P-TrCP chimera effectively reduced endogenous PD-1 levels in CAR-T cells.[000125] To investigate the functional impact of PD-1 degradation, P-TrCP chimera-modified CAR-T cells were co-cultured with SK-OV-3 cells stably expressing PD-L1 (SK-OV-3-PD-L1). At defined time points, CAR-T cells were harvested for enumeration and phenotypic analysis by flow cytometry. Compared with unmodified 4H1128z CAR-T cells, P-TrCP chimera-expressing CAR-T cells exhibited reduced PD-1 surface expression (Fig. 14G) and, notably, showed enhanced proliferation even in the presence of PD-L1 (Fig. 14H). These findings suggest that P- TrCP chimera-mediated PD-1 degradation mitigates PD-1 / PD-L1 -mediated proliferative suppression and may improve CAR-T cell persistence and function within the immunosuppressive tumor microenvironment (TME).Contingency Control: dTAG-mediated shutdown of PKO activity[000126] Having demonstrated the efficacy of PKO construct in degrading target proteins, it was next sought to incorporate a controllable safety mechanism that would allow rapid inactivation of PKO activity in the event of adverse effects during CAR-T therapy. Given the modular nature of PKO technology, the degradation tag (dTAG) system was identified as an ideal candidate for this purpose. dTAG employs heterobifunctional small molecules to recruit E3 ubiquitin ligases and selectively degrade proteins fused to the FKBP12F36Vdomain (as described by Behnam Nabet, et al. The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol. 2018 May; 14(5) :431-441. doi: 10.1038 / s41589-018-0021-8, which is incorporated herein by reference in its entirety). To enable external control of PKO activity, FKBP12F36V-PKO fusion constructs were generated by inserting the FKBP12F36V domain at the N terminus of both full-length and truncated P-TrCP.10GS.SH2(C) constructs (Fig. 15A).P-661940-PC[000127] The FKBP 12F36V-tagged PKO constructs were first overexpressed in HEK293T cells. Notably, the full-length 0-TrCP fusion protein was undetectable upon FKBP12F36Vfusion (Fig. 15B), suggesting possible instability or degradation. Therefore, only the truncated FKBP12F36V-0- TrCP(AWD).10GS.SH2(C) construct was used for subsequent experiments.[000128] To evaluate whether the PKO construct could be regulated by dTAG molecules, three compounds were tested: dTAG-7, dTAG-13, and dTAGV-1. dTAG-7 and dTAG-13 represent first-generation dTAG molecules that recruit the cereblon (CRBN) E3 ubiquitin ligase, though they differ in linker chemistry, which may influence their degradation efficiency (as described by Nabet et al. (2018), supra). In contrast, dTAGV-1 is a second-generation molecule that engages the von Hippel-Lindau (VHL) E3 ligase for target degradation (as described by Nabet, et al., Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat Commun. 2020 Sep 18; 11 (1 ):4687. doi: 10.1038 / s41467-020-18377-w, which is incorporated herein by reference in its entirety). To optimize degradation conditions, the FKBP12F36V-P- TrCP(AWD).10GS.SH2(C) construct (hereafter referred to as FKBP12F36V.PKO) was overexpressed in HEK293T cells and treated with each of the three dTAG molecules across multiple concentrations. dTAGV-1 and dTAG-13 exhibited optimal degradation at 0.1 pM, whereas dTAG-7 was most effective at 1 pM (Fig. 15C). Notably, increased concentrations did not further enhance degradation, underscoring the sensitivity and tunability of the dTAG system. In subsequent time-response assays, all three compounds induced time-dependent degradation of FKBP12F36V.PKO, with maximal effects observed at 24 hours (Fig. 15D). Based on these findings, it was established optimal working conditions as 0.1 pM for 24 hours for dTAGV-1 and dTAG-13, and 1 pM for 24 hours for dTAG-7.[000129] To assess the broader applicability of the dTAG-PKO platform, FKBP12F36V.PKO was tested in SH-SY5Y human neuroblastoma cells. Effective degradation of the construct was observed (Fig. 15E), indicating that the system is functional across diverse cellular contexts. Having established that our PKO constructs effectively degrade PD-1, next investigated was whether this degradation could be reversed using the dTAG system. To this end, PD-1 WT and FKBP12F36V.PKO were coexpressed in HEK293T cells for 36 hours, followed by treatment with each of the three dTAG molecules under their respective optimized conditions. As anticipated, degradation of PD-1 was reversed upon dTAG treatment, corresponding to the loss of FKBP12F36VPKO due to dTAG-mediated degradation (Fig. 15F). Interestingly, although allP-661940-PC three dTAG molecules efficiently degraded FKBP12F36V.PKO, recovery of PD-1 was more pronounced with the CRBN-recruiting dTAG-7 and dTAG- 13 than with the VHL-recruiting dTAGV-1. This observation suggests potential differences in the dynamics of E3 ligase engagement or downstream degradation efficiency and highlights an important consideration in selecting dTAG molecules for future applications.[000130] Taken together, the incorporation of the dTAG system enabled rapid and reversible control of PKO-mediated target degradation, thereby enhancing the flexibility and safety of the PKO platform for potential therapeutic and research applications.Discussion[000131] In this study, the regulatory roles of N-linked glycosylation in PD-1 protein stability were dissected, subcellular localization, and binding affinity with its ligand PD-L1. It was demonstrated that N-linked glycosylation plays a critical role in regulating PD-1 protein stability and membrane localization, consistent with findings by Sun et al. (supra). Four N-linked glycosylation sites — N49, N58, N74, and N116 — were identified and shown to cooperatively contribute to PD-1 stabilization. Although previous studies have reported that glycosylation at N58 is essential for PD-1 binding to PD-L1 (as described by J. Zhang et al., supra), the present data did not reveal a significant reduction in PD-L1 binding with the N58Q mutant, suggesting that under our experimental conditions, N58 glycosylation may not be strictly required for this interaction. Notably, all N-to-Q (NQ) mutants, with the exception of N49Q and N58Q, exhibited enhanced binding to PD-Ll-Fc compared to wild-type PD-1. These findings indicate that glycosylation at N74 and N116, rather than at N58, may serve a more inhibitory role in PD-L1 binding. Further investigation will be required to resolve these differences and clarify the context-dependent roles of individual glycosylation sites.[000132] The protein kinase GSK3P was found to regulate PD-1 stability (Figs. 8A-8H). Subsequently identified were three serine / threonine residues — SI 18, T120, and S127 — as potential sites of GSK3P-mediated phosphorylation. Interestingly, phosphorylation at SI 18 and T120 was found to be a prerequisite for N-linked glycosylation of PD-1, whereas S127 was largely dispensable for this modification (Fig. 8F). This phosphorylation-glycosylation crosstalk highlights a previously unrecognized regulatory axis in PD-1 posttranslational processing. A model is proposed in which newly synthesized PD-1 is initially phosphorylated by GSK3P andP-661940-PC then follows one of two distinct fates: (i) it is ubiquitinated by E3 ubiquitin ligase and targeted for proteasomal degradation, or (ii) it undergoes N-linked glycosylation, traffics to the plasma membrane, and engages PD-L1 to exert its immune checkpoint function.[000133] Targeted protein degradation (TPD) has emerged as a powerful strategy to eliminate disease-relevant proteins. While PROTACs are widely used, their reliance on ligandable pockets limits target scope. An alternative approach is presented: protein knockout (PKO), which employs a targeting peptide fused to an E3 ligase to induce direct, ligand-independent degradation of both nascent and existing proteins — offering rapid and efficient control of long-lived targets. Specifically, it was demonstrated that fusion of two SH2 domains from the SHP-2 phosphatase to the P-TrCP E3 ubiquitin ligase enabled efficient degradation of glycosylated PD-1. By targeting the cytosolic ITIM and ITSM motifs of PD-1, the 0-TrCP-SH2 chimera circumvented the protective effects of N-linked glycosylation.[000134] The PKO strategy offers several practical advantages over conventional gene editing and immunotherapy approaches. In contrast to complete gene knockout, engineered 0-TrCP chimeras reduce but do not entirely eliminate target protein levels. This partial depletion may reduce the severity of unintended consequences. For example, while CRISPR-mediated knockout of PD-1 in CAR-T cells significantly enhances antitumor efficacy, it also raises concerns regarding uncontrolled immune activation due to loss of PD-1 ’s immunoregulatory function (as described by O. O. Yeku, et al., supra). In contrast, PKO may provide a more balanced modulation of immune checkpoint signaling.[000135] PKO also streamlines CAR-T cell engineering by allowing simultaneous delivery of both the CAR construct and the PKO module within a single vector. This simplifies production compared to CRISPR-based methods, which require multiple components and delivery systems, or CAR-T cell therapies combined with anti-PD-1 antibodies, which involve separate treatment regimens and higher costs. The integrated CAR-PKO platform offers a unified, cost-effective solution that combines tumor targeting and checkpoint inhibition.[000136] Another major advantage of CAR-PKO is its ability to sustain PD-1 suppression autonomously. Prior studies, such as that by Cherkassky et al. Human CAR T cells with cell- intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J Clin Invest. 2016 Aug l;126(8):3130-44. doi: 10.1172 / JCI83092, demonstrated that PD-1 blockade can transientlyP-661940-PC reinvigorate exhausted CAR-T cells, but this effect is short-lived and requires repeated antibody administration. In contrast, CAR-PKO achieves continuous PD-1 degradation, potentially improving and prolonging CAR-T cell function within the immunosuppressive tumor microenvironment.[000137] To address safety concerns associated with constitutive protein degradation, PKO was enhanced further by integrating the dTAG system, which enables chemically inducible inactivation of PKO constructs. This feature provides an additional layer of control, allowing for rapid shutdown of degradation activity in the event of adverse effects, such as excessive immune activation (Figs. 15A-15F). Together, these features position PKO — and specifically CAR-PKO — as a flexible, efficient, and potentially safer alternative for next-generation cellular immunotherapies.[000138] Overall, the present findings reveal the regulatory roles of GSK3p-mediated phosphorylation and N-linked glycosylation in controlling PD-1 stability and function. More importantly, they highlight the potential of targeting PD-1 for degradation via the CAR-PKO strategy as a novel therapeutic approach in CAR-T cell-based cancer immunotherapy. Given its modular design, PKO can be adapted to target a wide range of intracellular proteins, offering a versatile platform to enhance the efficacy of CAR-T cell therapies.[000139] As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.[000140] In this disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” refers to one or more of such compounds and equivalents thereof known to one skilled in the art, and so forth. The term “plurality,” as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.[000141] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a firstP-661940-PC indicated number and a second indicated number and “ranging / ranges from” a first indicated number “to” a second indicated number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.[000142] When values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In one embodiment, the term “about” refers to a deviance of between 0.1-5% from the indicated number or range of numbers.[000143] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviations, per practice in the art. Alternatively, when referring to a measurable value such as an amount, a temporal duration, a concentration, and the like, may encompass variations of ±20% or ±10%, more specifically ±5%, even more particularly ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. In another embodiment, the term “about” refers to a deviance of between 1-10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of up to 20% from the indicated number or range of numbers. In one embodiment, the term “about” refers to a deviance of ± 10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of ± 5% from the indicated number or range of numbers.[000144] As used herein, the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” "treatment,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and / or physiologic effect by local and / or systemic action. A composition may include a therapeutic as described herein, e g., a CAR T-cell comprising a vector, said vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR and a second nucleic acid sequence encoding a chimeric complex comprising P-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein P-TrCP is a P-transducin repeat containing protein, and TP is a targetingP-661940-PC peptide that binds to PD-1 and is linked to the P-TrCP in a P-TrCP-containing SCF E3 ubiquitin ligase. In an embodiment, the TP is SH2 (C) and / or SH2 (N) domains of tyrosine phosphatase SHP2. In an embodiment, the TP is SH2 (C) (SEQ ID NO: 15) and / or SH2 (N) domains (SEQ ID NO: 16) of tyrosine phosphatase SHP2.[000145] As used herein, the terms “treatment” or “therapy” (as well as different forms thereof) include preventative (e.g., prophylactic), curative or palliative treatment. As used herein, the term “treating” includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease or disorder.[000146] The terms “subject,” “individual” and “patient” are used interchangeably herein, and refer to an animal, such as a human, to whom treatment, including prophylactic treatment, with a pharmaceutical composition described herein is provided. The term “subject” as used herein refers to human and non-human animals. The terms “non-human animals” and “non-human mammals” are used interchangeably and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens and turkeys.[000147] The therapeutic agents and pharmaceutical compositions comprising the therapeutic agents of the invention thus may include a “therapeutically effective amount.” A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.[000148] Furthermore, a skilled artisan would appreciate that the term “therapeutically effective amount” may encompass a total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.P-661940-PC[000149] The amount of a therapeutic agent of the invention that will be effective in the treatment of a particular disorder or condition, such as a cancer, including but not limited to a blood cancer or a solid tumor, in a human subject in need thereof, vary depending upon many different factors, also will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. In one embodiment, the pharmaceutical composition comprising the therapeutic agent, i.e., the CAR T-cell comprising the vector comprising the nucleic acid sequences described herein, is administered intravenously. In an embodiment, the pharmaceutical composition comprising the therapeutic agent, i.e., the CAR T-cell comprising the vector described herein, is administered intravenously and / or systemically. The pharmaceutical compositions containing the herein described therapeutic agent(s), may be administered only once, or it may be administered multiple times. For multiple dosages, the pharmaceutical composition may be, for example, administered three times a day, twice a day, once a day, once every two days, twice a week, weekly, once every two weeks, or monthly.[000150] All patents and literature references cited in the present specification are hereby incorporated by reference in their entirety.[000151] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration. It is to be understood that the invention is not limited to the precise embodiments, and that various modifications, substitutions, changes, and equivalents may be effected therein by those of ordinary skill in the art without deviating from the scope of the invention as defined in the appended claims.

Claims

P-661940-PCWHAT IS CLAIMED IS:

1. A chimeric complex of 0-TrCP-TP SCF (Skpl-Cullinl-F-box) E3 ubiquitin ligase, wherein -TrCP is a 0-transducin repeat containing protein, and TP is a targeting peptide that binds to PD- 1 and is linked to the -TrCP in a -TrCP -containing SCF E3 ubiquitin ligase.

2. The chimeric complex of claim 1, wherein the TP is SH2 (C) (SEQ ID NO:15) and / or SH2 (N) domains (SEQ ID NO: 16) of Tyrosine phosphatase SHP2.

3. A vector comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and a second nucleic acid sequence encoding the chimeric complex of claim 1 or 2, wherein the nucleic acid encoding the chimeric complex comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or the nucleic acid sequence set forth in SEQ ID NO: 3.

4. CAR-T cells transduced by the vector of claim 3.

5. A method for increasing the expansion of CAR T-cells, comprising the steps of: transducing T cells with the vector of claim 3, selectively depleting glycosylated PD-1, and enhancing the ex vivo expansion of CAR T cells.

6. A method for improving CAR-T cell effector functions, comprising the steps of: transducing T cells with the vector of claim 3, thereby selectively depleting glycosylated PD-1, enhancing the ex vivo expansion of CAR T cells, and improving the CAR-T cell effector functions.

7. A method for in vivo expansion of CAR T-cells in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting 0-TrCP chimeric complex, said complex comprising -TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein the immune checkpoint protein / receptor is selectively depleted.P-661940-PC8. The method of claim 7, wherein the immune checkpoint protein / receptor-targeting P-TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:3.

9. The method of claim 7, wherein the chimeric antigen receptor (CAR) is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1.

10. The method of claim 7, wherein the linker is 10GS, and the 10GS encoded by a nucleic acid sequence set forth in SEQ ID NO: 10.

11. The method of claim 7, wherein the TP or fragment of the TP binds to the immune checkpoint immune checkpoint protein / receptor, and the immune checkpoint immune checkpoint protein / receptor is PD-1, and the PD-1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 11.

12. The method of claim 7, wherein the endogenous PD-1 is on CAR-T cells and is glycosylated.

13. The method of claim 7, wherein the endogenous PD-1 on CAR-T cells and is nonglycosylated.

14. The method of claim 7, wherein the TP is a SH2 (C) domain of tyrosine phosphatase SHP2, and the SH2 (C) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO:15.

15. The method of claim 7, wherein the TP is a SH2 (N) domain of tyrosine phosphatase SHP2, and the SH2 (N) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO:16.

16. The method of claim 7 , wherein the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 5 and a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 6.

17. The method of claim 7, wherein the CAR comprises an extracellular (EC) antigen recognition domain (SEQ ID NO:4), a transmembrane (TM) domain (SEQ ID NO:5) and an intracellular (IC) signaling domain (SEQ ID NO: 6).P-661940-PC18. A method for selectively degrading endogenous PD-1 in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting 0-TrCP chimeric complex, said complex comprising 0-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein expression of the 0-TrCP chimeric complex selectively degrades endogenous PD-1.

19. The method of claim 18, wherein the immune checkpoint protein / receptor-targeting 0- TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

20. The method of claim 18, wherein the chimeric antigen receptor (CAR) is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1.

21. The method of claim 18, wherein the linker is 10GS, and the 10GS encoded by a nucleic acid sequence set forth in SEQ ID NO: 10.

22. The method of claim 18, wherein the TP or fragment of the TP binds to the immune checkpoint immune checkpoint protein / receptor, and the immune checkpoint immune checkpoint protein / receptor is PD-1, and the PD-1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 11.

23. The method of claim 18, wherein the endogenous PD-1 is on CAR-T cells and is glycosylated.

24. The method of claim 18, wherein the endogenous PD-1 on CAR-T cells and is nonglycosylated.

25. The method of claim 18, wherein the TP is a SH2 (C) domain of tyrosine phosphatase SHP2, and the SH2 (C) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15.

26. The method of claim 18, wherein the TP is a SH2 (N) domain of tyrosine phosphatase SHP2, and the SH2 (N) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQP-661940-PCID NO: 16.

27. The method of claim 18, wherein the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16.

28. The method of claim 18, wherein the CAR comprises an extracellular (EC) antigen recognition domain (SEQ ID NO:4), a transmembrane (EM) domain (SEQ ID NO:5) and an intracellular (IC) signaling domain (SEQ ID NO: 6).

29. A method for enhancing efficacy and anti-tumor activity of a CAR T-cell in a subject in need thereof, the method comprising administering a therapeutically effective amount of a CAR T-cell to the subject, wherein the CAR T-cell comprises a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen and (b) a nucleic acid sequence encoding an immune checkpoint protein / receptor-targeting P-TrCP chimeric complex, said complex comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP, wherein expression of the CAR and the P-TrCP chimeric complex in the subject enhances the efficacy and anti-tumor activity of the CAR T-cell.

30. The method of claim 29, wherein the immune checkpoint protein / receptor-targeting P- TrCP chimeric complex is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

31. The method of claim 29, wherein the chimeric antigen receptor (CAR) is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1.

32. The method of claim 29, wherein the linker is 10GS, and the 10GS encoded by a nucleic acid sequence set forth in SEQ ID NO: 10.

33. The method of claim 29, wherein the TP or fragment of the TP binds to the immune checkpoint immune checkpoint protein / receptor, and the immune checkpoint immune checkpoint protein / receptor is PD-1, and the PD-1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 11.

34. The method of claim 33, wherein the P-TrCP chimeric complex selectively degrades endogenous PD-1.P-661940-PC35. The method of claim 29, wherein the endogenous PD-1 is on CAR-T cells and is glycosylated.

36. The method of claim 29, wherein the endogenous PD-1 on CAR-T cells and is nonglycosylated.

37. The method of claim 29, wherein the TP is a SH2 (C) domain of tyrosine phosphatase SHP2, and the SH2 (C) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15.

38. The method of claim 29, wherein the TP is a SH2 (N) domain of tyrosine phosphatase SHP2, and the SH2 (N) domain of SHP2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 16.

39. The method of claim 29, wherein the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16.

40. The method of claim 29, wherein the CAR comprises an extracellular (EC) antigen recognition domain (SEQ ID NO:4), a transmembrane (EM) domain (SEQ ID NO:5) and an intracellular (IC) signaling domain (SEQ ID NO: 6).

41. A method for deactivating or reversing degradation of a target protein by CAR T-cells in vivo in a subject treated with an anti -tumor CAR T-cell, wherein the target protein is PD-1, the method comprising: a) inserting a nucleic acid encoding a FKBP12F36Vdomain (SEQ ID NO: 17) at the N terminus of a nucleic acid encoding a PD-1 targeting 0-TrCP chimeric complex (SEQ ID NO: 2 or SEQ ID NO:3) comprising P-TrCP E3 ubiquitin ligase fused via a linker to a targeting peptide (TP) or fragment of the TP to form a PD-1 targeting FKBP12F36V-P-TrCP chimeric complex; b) transducing the anti-tumor CAR T-cell with a vector comprising (a) a nucleic acid sequence encoding a chimeric antigen receptor (“CAR”) targeting a tumor antigen (SEQ ID NO: 1) and (b) a nucleic acid sequence encoding the PD-1 targeting FKBP12F36V-0-TrCP chimeric complex;P-661940-PC c) administering a therapeutically effective amount of the anti-tumor CAR T-cell comprising the a nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding PD- 1 targeting FKBP12F36V-P-TrCP chimeric complex to a subject in need thereof to express the CAR and the PD-1 targeting FKBP12F36V-P-TrCP chimeric complex in the subject, wherein expression of the CAR and the FKBP12F36V-P-TrCP chimeric complex in the subject enhances the efficacy and anti-tumor activity of the CAR T-cell; and d) administering a degradation tag (dTAG) molecule to the subject to degrade the PD-1 targeting P-TrCP chimeric complex, thereby deactivating or reversing degradation of PD- 1 to alleviate or reduce adverse effects of the degradation of PD-1.

42. The method of claim 41, wherein the dTAG molecule is dTAG-7, dTAG-13, or dTAGV- 1. dTAG-7 and dTAG-13.

43. A retroviral CAR construct comprising a nucleic acid encoding an anti-tumor CAR, a nucleic acid encoding P-TrCP chimera, and a nucleic acid encoding a self-cleaving 2A peptide derived from porcine teschovirus (P2A) linking the CAR and the P-TrCP chimera, wherein said CAR construct simultaneously expresses the upstream CAR and the downstream P-TrCP chimera.

44. The retroviral CAR construct of claim 43, wherein the nucleic acid encoding the selfcleaving 2A peptide is set forth in SEQ ID NO: 9.

45. The retroviral CAR construct of claim 43, wherein the encoded P-TrCP chimera is a full- length P-TrCP protein encoded by the nucleic acid set forth in SEQ ID NO: 2.

46. The retroviral CAR construct of claim 43, wherein the encoded P-TrCP chimera is a truncated P-TrCP protein lacking the WD40 domain (“P-TrCP (AWD)”), said P-TrCP chimera is encoded by the nucleic acid set forth in SEQ ID NO: 3.

47. The retroviral CAR construct of claim 43, wherein the TP comprises a SH2 (C) domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and / or a SH2 (N) domain of tyrosine phosphatase SHP2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16.

48. The retroviral CAR construct of claim 43, wherein the P-TrCP chimera is P- TrCP.10GS.SH2(C), P-TrCP.10GS.SH2(N), P-TrCP(AWD).10GS.SH2(C), or P- TrCP(AWD).1 OGS . SH2(N).P-661940-PC49. The retroviral CAR construct of claim 43, wherein the 0-TrCP chimera is 0- TrCP.1 OGS. SH2(C), 0-TrCP.1 OGS. SH2(N).

50. The retroviral CAR construct of any one of claims 43-49, further comprising a FKBP12F36Vdomain (SEQ ID NO: 17) at the N-terminus of the nucleic acid encoding the 0-TrCP chimera, wherein the nucleic acid encoding the 0-TrCP chimera is set forth in SEQ ID NO: 2 or SEQ ID NO:3.

51. The retroviral CAR construct of any one of claims 43-49, a nucleic acid encoding an antitumor CAR is set forth in SEQ ID NO: 1.

Images