Rejuvenation of CAR T cells

Abstract

Provided herein is a drug payload conjugated with a targeting ligand that is specifically designed to be delivered to exhausted CAR T cells to rejuvenate them. The targeted CAR T cells are engineered with a fusion receptor that can bind to the targeting ligand and internalize the conjugated drug payload to perform its regulatory function on the exhausted CAR T cells.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure provides a system for rejuvenating chimeric antigen receptor T (CAR T) cells exhausted by cancer antigens. In particular, the system includes a fusion receptor in a classical CAR construct that provides a ligand-binding module that recognizes high affinity ligand-payload drug conjugates and delivers a drug payload designed to block inhibitory signaling of exhausted CAR T or reactivate CAR T via an antigen-independent pathway. [Background technology]

[0002] background The field of chimeric antigen receptor (CAR) T-cell therapy has made considerable progress over the past two decades. CAR constructs consist of four parts: (1) an extracellular binding moiety for tumor-specific antigens, (2) a hinge domain, (3) a transmembrane domain, and (4) a combination of various activation domains, such as CD28, 4-1BB, and CD3ζ chain. The most impressive success has been seen in CAR T therapy for CD19-positive B-cell leukemia, where complete remission rates of over 80% have been achieved in several clinical trials.

[0003] In contrast, treating solid tumors with CAR T has proven challenging to date. There has been great success in preclinical studies using syngeneic mouse models or in xenograft tumor models in immunodeficient mice. However, none of the clinical trials involving solid tumors, e.g., breast, ovarian, or lung cancer, have shown improvement compared to controls. In general, CAR T suffers from the same constraints faced by other adoptive cell therapies, including: (1) insufficient tumor penetration; (2) low oxygen tension; and (3) an immunosuppressive tumor microenvironment that includes tumor-associated macrophages, fibroblasts, and inhibitory cytokines. 1CAR T cells, like tumor-infiltrating lymphocytes (TILs), can be reeducated by this suppressive microenvironment and transition into a "hypofunctional" state characterized by overexpression of co-inhibitory molecules (i.e., PD-1, Tim-3, LAG-3, etc.), INF-γ secretion, and reduced killing capacity. Data from patient ovarian cancer samples suggest that PD-1 + CD8 + The majority of T cells did not express CD127, which is known to be important for effector-memory transition in T cells. LAG-3 overexpression also promoted TCR-specific CD8 + It was negatively correlated with the effector function of TILs. + LAG-3 + Double positive T cells showed less INFγ production 2 Similarly, NY-ESO-1 TCR-specific human T cells became hypofunctional in mouse solid tumor models, exhibiting high expression of co-inhibitory molecules and inefficient antitumor activity, which was attributed to the microenvironment and constant T cell activation due to continuous exposure to antigen. 3 Therefore, for better treatment of solid tumors, it is highly desirable to reverse the suppressive microenvironment and, more importantly, to rejuvenate exhausted CAR T cells. Summary of the Invention

[0004] The present disclosure provides a system for rejuvenating exhausted classical CAR T cells. The system includes at least two components, the first component is a conjugate that includes a targeting ligand covalently linked to a payload drug, and the second component is a targeting ligand binding module linked to a membrane anchor module. The targeting ligand binding module of the second component recognizes the targeting ligand in the first component with high affinity to form a complex, and the payload drug blocks the inhibitory signaling of exhausted classical CAR T or reactivates the CAR T through an antigen-independent pathway. The membrane anchor module mediates the internalization of the two-component complex into exhausted CAR T cells.

[0005] In some preferred embodiments, the targeting ligand of the first component is folic acid, FITC, or FK506.

[0006] In some preferred embodiments, the target ligand binding module of the second component comprises a folate receptor, an anti-FITC antibody fragment, or an FKBP.

[0007] In some preferred embodiments, the membrane anchor module is a folate receptor.

[0008] In some preferred embodiments, the first component comprises a releasable linker between the targeting ligand and the payload drug.

[0009] In some preferred embodiments, the first component comprises a non-releasable linker between the targeting ligand and the payload drug.

[0010] In some preferred embodiments, the binding affinity between the target ligand and the target ligand binding module is in the sub-nM range.

[0011] In some preferred embodiments, the drug payload is a Toll-like receptor 7 (TLR7) agonist or a Simulator of interferon genes (STING) agonist.

[0012] In some preferred embodiments, the drug payload is an inhibitor against the following proteins: SHP1 / 2, TC-PTP or DGKα, TGFβ.

[0013] In some preferred embodiments, the TLR7 agonist is It has the structure TIFF0007623935000001.tif56134.

[0014] In some preferred embodiments, the first component is It is a fluorescein-TLR7 agonist having the structure TIFF0007623935000002.tif49128.

[0015] In some preferred embodiments, the first component is FK506-TLR7 agonist with structure TIFF0007623935000003.tif52129.

[0016] In some preferred embodiments, the first component is selected from the group consisting of: It is one of the files: TIFF0007623935000004.tif60165.

[0017] In some preferred embodiments, the first component is a TC-PTP phosphatase inhibitor, such as: TIFF0007623935000005.tif23144.

[0018] In some preferred embodiments, the phosphatase inhibitor has the following structure: It is linked to fluorescein or FK506 (tacrolimus) to form TIFF0007623935000006.tif80128.

[0019] In some preferred embodiments, the payload drug in the first component has the following structure: TIFF0007623935000007.tif51128 containing one STING agonist.

[0020] In some preferred embodiments, the first component has the following structure between the targeting ligand and the payload drug: TIFF0007623935000008.tif74157.

[0021] The present disclosure further provides a method for rejuvenating exhausted CAR T cells, the method comprising: (a) providing to the exhausted CAR T cells a first component comprising a conjugate, the conjugate comprising a targeting ligand covalently linked to a drug payload via a releasable or non-releasable linker; (b) providing to the exhausted CAR T cells a second component comprising a fusion receptor linked to an exhausted CAR construct, the fusion receptor comprising a targeting ligand binding module and a membrane anchor module; (c) binding the target ligand binding module of the second component to the target ligand in the first component to form a complex; (d) allowing the membrane anchor module to mediate internalization of the complex into the exhausted CAR T cells; (e) Allowing the payload drug to block inhibitory signaling of exhausted CAR T or reactivate said CAR T via an antigen-independent pathway. Includes.

[0022] In some preferred embodiments, the above methods deliver a payload drug that performs its function in the endosomes of the exhausted CAR T, and the targeting ligand and the payload drug are linked by a non-releasable linker.

[0023] In some preferred embodiments, the above methods deliver a payload drug that performs its function as a free drug in the cytosol of the exhausted CAR T, and the targeting ligand and the payload drug are linked by a releasable linker.

[0024] In some preferred embodiments, in the above methods, the targeting ligand of the first component is folate, FITC, or FK506.

[0025] In some preferred embodiments, in the above methods, the target ligand binding module of the second component is anti-FITC, folate receptor, or FKBP.

[0026] In some preferred embodiments, in the above methods, the target ligand binding module of the second component is folate receptor alpha (FRa).

[0027] In some preferred embodiments, in the above methods, the drug payload of the first component is a Toll-like receptor 7 (TLR7) agonist or a stimulator of interferon genes (STING) agonist.

[0028] In some preferred embodiments, in the above methods, the drug payload of the first component is an inhibitor against the following proteins: SHP1 / 2, TC-PTP or DGKα, TGFβ.

[0029] In some preferred embodiments, in the above methods, the TLR7 agonist is TIFF0007623935000009.tif has a structure of 55133.

[0030] In some preferred embodiments, in the above method, the first component comprises: It is a fluorescein-TLR7 agonist having the structure TIFF0007623935000010.tif49128.

[0031] In some preferred embodiments, in the above method, the first component comprises: It is an FK506-TLR7 agonist with the structure TIFF0007623935000011.tif52129.

[0032] In some preferred embodiments, in the above method, the first component comprises: The file is TIFF0007623935000012.tif46143.

[0033] In some preferred embodiments, in the above methods, the first component is a TC-PTP phosphatase inhibitor: TIFF0007623935000013.tif23144.

[0034] In some preferred embodiments, in the above methods, the first component has the following structure: The antibody contains a phosphatase inhibitor linked to fluorescein or FK506 (tacrolimus) to form TIFF0007623935000014.tif83128.

[0035] In some preferred embodiments, in the above methods, the first component has the following structure: TIFF0007623935000015.tif51128 contains one STING agonist payload drug.

[0036] In some preferred embodiments, in the above method, the first component has the following structure between the targeting ligand and the payload drug: TIFF0007623935000016.tif74157.

[0037] [The present invention 1001] 1. A system for rejuvenating exhausted classical CAR T cells comprising at least two components, wherein a first component is a conjugate comprising a targeting ligand covalently linked to a payload drug, and a second component is a targeting ligand binding module linked to a membrane anchor module, wherein the targeting ligand binding module of the second component recognizes the targeting ligand in the first component with high affinity to form a complex, wherein the payload drug blocks inhibitory signaling of the exhausted classical CAR T or reactivates the CAR T via an antigen-independent pathway, and the membrane anchor module mediates internalization of the two-component complex into the exhausted CAR T cells. [The present invention 1002] The system of the present invention 1001, wherein the targeting ligand of the first component is folic acid, FITC, or FK506. [The present invention 1003] The system of the present invention 1001, wherein the target ligand binding module of the second component is an anti-FITC antibody fragment, FKBP, or a folate receptor. [The present invention 1004] The system of the present invention 1001, wherein the membrane anchor module is a folate receptor. [The present invention 1005] The system of the present invention 1001, wherein the first component comprises a releasable linker between the targeting ligand and the payload drug. [The present invention 1006] The system of the present invention 1001, wherein the first component comprises a non-releasable linker between the targeting ligand and the payload drug. [The present invention 1007] The system of the present invention 1001, wherein the binding affinity between the target ligand and the ligand binding module is in the sub-nM range. [The present invention 1008] The system of the present invention 1001, wherein the drug payload is a Toll-like receptor 7 (TLR7) agonist or a Simulator of interferon genes (STING) agonist. [The present invention 1009] The system of the present invention 1001, wherein the drug payload is an inhibitor against the following proteins: SHP1 / 2, TC-PTP or DGKα, TGFβ. [The present invention 1010] TLR7 agonists, TIFF0007623935000017.tif55133 The system of the present invention 1008 has the following structure. [The present invention 1011] The first component is TIFF0007623935000018.tif49128 The system of the present invention 1001 is a fluorescein-TLR7 agonist having the structure: [The present invention 1012] The first component is TIFF0007623935000019.tif51129 The system of the present invention 1001 is an FK506-TLR7 agonist having the structure: [The present invention 1013] The first component is: TIFF0007623935000020.tif60165 The system of the present invention 1001 is one of the above. [The present invention 1014] The first component is a TC-PTP phosphatase inhibitor: TIFF0007623935000021.tif23144 The system of the present invention 1001, comprising a payload drug selected from the group consisting of: [The present invention 1015] The phosphatase inhibitor has the following structure: TIFF0007623935000022.tif78128 The system of the present invention 1014, wherein the compound is linked to fluorescein or FK506 (tacrolimus) to form [The present invention 1016] The payload drug in the first component has the following structure: TIFF0007623935000023.tif51128 The system of the present invention 1001, comprising one of the STING agonists. [The present invention 1017] The first component has, between the targeting ligand and the payload drug, the following structure: TIFF0007623935000024.tif74161 The system of the present invention 1001, comprising a spacer selected from the group consisting of: [The present invention 1018] 1. A method for rejuvenating exhausted CAR T cells, comprising: (a) providing to the exhausted CAR T cells a first component comprising a conjugate, the conjugate comprising a targeting ligand covalently linked to a drug payload via a releasable or non-releasable linker; (b) providing to the exhausted CAR T cells a second component comprising a fusion receptor linked to an exhausted CAR construct, the fusion receptor comprising a targeting ligand binding module and a membrane-bound receptor module; (c) binding the target ligand binding module of the second component to the target ligand in the first component to form a complex; (d) allowing the membrane-bound CAR module to mediate internalization of the complex into the exhausted CAR T cells; (e) Allowing the payload drug to block inhibitory signaling of exhausted CAR T or reactivate the CAR T via an antigen-independent pathway. The method comprising: [The present invention 1019] The method of the present invention 1018, wherein the payload drug performs its function in the endosomes of the exhausted CAR T, and the targeting ligand and the payload drug are linked by a non-releasable linker. [The present invention 1020] The method of the present invention 1018, wherein the payload drug performs its function as a free drug in the cytosol of the exhausted CAR T, and the targeting ligand and the payload drug are linked by a releasable linker. [The present invention 1021] The method of any one of claims 10 to 18, wherein the targeting ligand of the first component is folic acid, FITC, or FK506. [The present invention 1022] The method of any one of claims 10 to 18, wherein the target ligand binding module of the second component is anti-FITC, folate receptor, or FKBP. [The present invention 1023] The method of any one of claims 10 to 18, wherein the ligand binding module is folate receptor alpha (FRa). [The present invention 1024] The method of claim 1018, wherein the drug payload is a Toll-like receptor 7 (TLR7) agonist or a stimulator of interferon genes (STING) agonist. [The present invention 1025] The method of claim 1018, wherein the drug payload is an inhibitor against the following proteins: SHP1 / 2, TC-PTP or DGKα, TGFβ. [The present invention 1026] TLR7 agonists, TIFF0007623935000025.tif56133 The method of the present invention 1018, having the structure: [The present invention 1027] The first component is TIFF0007623935000026.tif49128 The method of the present invention, wherein the compound is a fluorescein-TLR7 agonist having the structure: [The present invention 1028] The first component is TIFF0007623935000027.tif51129 The method of the present invention, wherein the FK506-TLR7 agonist has the structure: [The present invention 1029] The first component is: TIFF0007623935000028.tif60165 The method of the present invention 1018 is one of the above. [The present invention 1030] The first component is a TC-PTP phosphatase inhibitor: TIFF0007623935000029.tif23144 The method of claim 1018, further comprising administering to said patient a payload drug selected from the group consisting of: [The present invention 1031] The phosphatase inhibitor has the following structure: TIFF0007623935000030.tif78128 The method of claim 10, wherein the compound is linked to fluorescein or FK506 (tacrolimus) to form [The present invention 1032] The payload drug in the first component has the following structure: TIFF0007623935000031.tif51128 The method of the present invention, comprising administering to the patient a STING agonist selected from the group consisting of: [The present invention 1033] The first component has, between the targeting ligand and the payload drug, the following structure: TIFF0007623935000032.tif77162 The method of claim 1018, further comprising a spacer selected from the group consisting of: These and other features, aspects, and advantages of the present invention will become better understood with reference to the following drawings, associated description, and claims. [Brief description of the drawings]

[0038] [Figure 1] Figure 1a: Graphical representation of the exhaustion model. Figure 1b: After three stimulations with fresh Raji cells in vitro, anti-CD19 CAR T cells become exhausted, as shown by a reduced killing activity. [Diagram 2] Graph of the secret passageway: FKBP or anti-FITC is linked to FRa as a fusion receptor, which continuously internalizes and delivers a payload linked to FK506 or FITC into the cell. [Diagram 3] Binding affinity of FK506 and FITC to the corresponding fusion receptors. FK506-rhodamine shows Kd=3.39 nM to the FKBP-FR fusion receptor. In contrast, FITC-AF647 shows Kd=8.03 nM to anti-FITC-FR. [Figure 4] Figure 4a: Structure of TLR7 agonists. Figure 4b: Rejuvenating effect of TLR7 agonists on exhausted anti-CD19 CAR T cells shown as killing activity, increased INFr levels, and decreased levels of co-inhibitory molecules. [Diagram 5] Chemical structures of TLR7 agonists. [Figure 6]In vitro model for inducing CAR T cell exhaustion. (A) CD19+ Raji cells were co-cultured with anti-CD19 CAR T cells at a 1:1 ratio, and fresh Raji cells were added every 12 hours. (B) With increasing number of stimulations (number of Raji cell additions), the lytic effect of CAR T cells gradually decreased. CD19-K562 cells were used as a control. (C) Expression level changes of co-inhibitory molecules PD-1, LAG3, and Tim3 targeting stim1 and stim3 in CD4- and CD8-positive CAR T cells. [Figure 7] Assessment of the effect of TLR7 agonists and PTP1b inhibitors on the rejuvenation of exhausted CAR T cells. (A) Chemical structures of TLR7 agonists and PTP1b inhibitors. (B-C) Exhausted CAR T cells were incubated with different concentrations of TLR7 agonists and PTP1b inhibitors and monitored by lytic effect and INFγ (B) as well as expression levels of PD-1, LAG-3, and Tim3 after incubation (C). * indicates p-value <0.05, ** indicates <0.01. ns = not significant. [Figure 8] Evaluation of potential derivatization sites of TLR7 agonists for targeted delivery by non-releasable ligands. (A) Chemical structures of TLR7 agonist analogs. (B) Exhausted CAR T cells were incubated with different concentrations of TLR7 analogs and the lytic effect was measured and compared to untreated groups. [Figure 9]Design and evaluation of releasable and non-releasable targeted delivery of TLR7 agonists using FITC as a targeting ligand. (A) Diagram of the chemical structure of FITC-TLR7 agonists. (B) Exhausted CAR T cells were incubated with different concentrations of releasable FITC-TLR7 and non-releasable FITC-JTLR7 agonists and the lytic effect was measured and compared to the untreated group. * indicates p-value <0.05, ** indicates <0.01. ns = not significant. (C) Left, crystal structure of FITC (green) bound to FITC scFv (grey) (PDB:1X9Q, left). The distance between FITC and the edge of FTIC scFv was measured to be about 18 Å. Right, similarly, crystal structure of R-848 (red) bound to TLR7 (grey) (PDB:5GMF) is shown. The distance between R-848 and the edge of TLR7 was measured to be about 24 Å. (D) Diagram showing two possible mechanisms of action for non-releasable FITC-TLR7 agonists: "Reaching" or "Jumping" modes. [Figure 10] Chemical linkers of various rigidity and hydrophobicity can be used in the design of targeted ligand-payload conjugates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description Although the concepts of the present disclosure have been illustrated and described in detail in the drawings and description herein, the results in the drawings and description should be considered as illustrative and not restrictive in character, it being understood that only exemplary embodiments have been shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0040] Unless otherwise defined, scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0041] Recently, chimeric antigen receptor (CAR) T-cell therapy has achieved great success in treating several types of hematopoietic cancers. On the other hand, it must be recognized that even with CAR T-cell therapy, some lymphomas and most solid tumor cases have very low response rates or high relapse rates. This is mainly due to one or a combination of the following three reasons: 1. emergence of antigen-negative cancer cell colonies under the selective pressure of CAR T cells, as observed in the case of CD19-negative ALL relapse treated with anti-CD19 CAR T cells; 2. aberrant tumor vasculature, dense stromal barrier, and suppressive microenvironment impede the initial homing and proliferation of CAR T cells in solid tumors; and 3. gradual exhaustion of CAR T cells after continuous exposure to tumor antigens, resulting in a decrease in their lytic efficacy. Given the absence of antigen loss in certain solid tumor patients (confirmed by biopsy sampling), the possible failure of CAR T-cell therapy is very likely due to the last two reasons. Therefore, practical methods to evaluate and rejuvenate CAR T cells in vivo to enhance CAR T cell efficacy in solid tumors are highly desirable.

[0042] Herein, we describe the novel design of a private passageway fusion receptor in CAR T cells as a universal platform to serve both purposes. This FITC-FR fusion receptor is composed of two parts, an scFv against FITC, a ligand-binding domain at the N-terminus, and a GPI anchor-internalization domain, FRα, at the C-terminus. To overcome the exhaustion state of CAR T cells in the suppressive tumor microenvironment, the FITC-FR fusion receptor can be specifically targeted by FITC-immunoagonists when expressed independently on CAR T cells. These immunoagonists, which usually cause strong autoimmune side effects, can now be administered systemically in a FITC-targeted form and safely delivered to FITC-FR-positive CAR T cells. In the past decades, great progress has occurred in the field in terms of cell types, delivery methods, and appropriate disease models. In terms of cell types, current cell therapies can be broadly categorized as chimeric antigen receptors (CARs), cells for tumor models, and stem cell-based regenerative medicine.

[0043] CAR T, also known as chimeric T cell receptor, chimeric immune receptor, or artificial T cell receptor, allows immune effector cells (usually T cells or NK cells) to recognize target cells bearing the corresponding antigen and exert cytotoxic activity. The emergence and development of CAR-T technology offers promise for certain types of cancer, making CAR-T a superstar in both biomedical and clinical research. Several conventional and improved CAR T cell designs are disclosed in U.S. Application Serial No. 15 / 296,666, the contents of which are incorporated herein in their entirety. In the '666 application, the CAR system is generated by providing cytotoxic lymphocytes expressing a CAR that targets a moiety not produced or expressed by the cells of the subject being treated. Thus, the CAR system allows the cytotoxic lymphocytes to be concentrated and targeted to target cells, such as cancer cells. The targeting moiety is part of a small conjugate molecule (SCM) that also contains a tumor cell receptor ligand. Upon administration of SCM and CAR-expressing cytotoxic lymphocytes, the cytotoxic lymphocyte response is targeted only to cells expressing the tumor receptor to which SCM binds.

[0044] Despite the rapid progress of CAR T cell therapy in both research and clinical applications, there are concerns associated with CAR T therapy. One of the fatal side effects is a cytokine storm resulting from the rapid lysis of tumor cells, which kills CAR-bearing normal cells. To address such side effects, PCT / US2018 / 018557 has developed targeted delivery of CAR T cells and specific drug payloads to target tumor cells to control such side effects. The contents of PCT / US2018 / 018557 are incorporated herein in their entirety. Briefly, an engineered protein is conjugated with a high-affinity targeting ligand. In this case, the targeting ligand carries at least one drug payload that is internalized by CAR T cells via the engineered protein to modulate the effect of transplanted cell therapy.

[0045] Another limitation of CAR T therapy is its tendency to become exhausted after repeated stimulation with cancer antigens. Re-stimulation after an overnight withdrawal from antigen ("rest") has been shown to result in reversibility of the exhausted T cell phenotype, as T cells isolated from solid tumor tissues exhibit high levels of INFγ secretion and killing activity. 4 However, it would be attractive to rejuvenate T cells in a more clinically relevant way using drugs that block inhibitory signaling or activate T cells through other pathways. Antibodies targeting checkpoint inhibitors (i.e., PD-1, CTLA-4, etc.) have shown some success in the clinic and in solid tumors. However, a combination of two or more targets often proves necessary. 5 6 In addition, antibody therapy has the disadvantage of poor penetration into solid tumors. Therefore, there have been few reports of combination therapy of CAR T and antibodies against checkpoint inhibitors in solid tumors. 7 and TC-PTP 8 Inhibition of phosphatases such as , is another approach to block inhibitory pathways. Both knockout experiments and small molecule inhibitors of these phosphatases have shown potent effects on lowering TCR thresholds and increasing T cell activity, but neither has been used in CAR T therapy. DGKα is another physiological inhibitor of TCR signaling and is overexpressed in exhausted TILs. DGKα catabolizes DAG to PA, thereby decreasing DAG levels and thus attenuating Ras and MARK ERK signaling. DGKα inhibitors restore degranulation and increase the killing activity of TILs and CAR Ts. 3 9 Another approach to rejuvenate T cells is to activate them through antigen-independent pathways. It has been shown that certain pathogen pattern recognition (PPR) receptors, including Toll-like receptors (TLRs), are expressed on non-myeloid cell populations, including T cells, and can be activated in the same way. Studies have shown that TLR2 10 , 4 11 , and 7 / 8 12 13Agonists have also been shown to activate CD8 T cells and increase INFγ secretion. However, the potent side effects of systemic administration of TLR agonists have 14 However, none of these agonists have been used in CAR T therapy to reactivate T cells or alter the immunosuppressive microenvironment, which is hindered by the controversial effects of TLR agonists on the tumor cells themselves. 15 Stimulator of interferon genes (STING) is a cytosolic DNA sensor (CDS) that is widely expressed in hematopoietic cells in peripheral lymphoid tissues, including T cells, myeloid cells, and monocytes. STING agonists have been used as immune stimulants in many immunotherapies and may also have a very significant impact in CAR T therapy. However, although the described inhibitors and agonists may have a very significant rejuvenating effect on CAR T cells, they are also very likely to induce significant side effects when administered systemically due to their very strong pro-inflammatory functions. Therefore, targeted delivery of potential payloads to CAR T cells is highly desirable.

[0046] To solve the specific delivery problem, we designed a secret pathway platform that allows a certain payload to be administered systemically and expressed in T cells together with the CAR construct so that it can accumulate specifically only in the CAR T cells while leaving other cells unchanged. The system consists of a fusion receptor and a classical CAR construct linked via a T2A self-cleavable sequence. The fusion receptor contains two parts: (1) a ligand-binding module that can recognize high affinity ligand-payload conjugates, and (2) a membrane-bound receptor module that can mediate internalization of the receptor / conjugate complex into cells. Two protein / ligand pairs, FKBP / FK506 and scFv(4M5.3) / FITC against FITC, were selected for part 1 because: (1) the absence of FKBP or 4M5.3 on the native cell membrane ensures specific delivery of the payload to fusion receptor-positive CAR T cells, thus reducing side effects on other cells, and (2) the sub-nanomolar binding affinity between the protein / ligand pairs promotes sufficient payload accumulation in the target cells. For membrane-bound receptors in part 2, folate receptor α (FRa) was selected due to its constitutive internalization property independent of folate binding. 16 We also designed a target-payload conjugation system, in which the payload can be linked to the targeting ligand using a non-releasable linker or a releasable disulfide linker depending on the target. Specifically, since TLR7 is in the endosome, the TLR7 agonist delivered by the covert pathway can exert its function as soon as it enters the endosome by receptor-mediated internalization. Therefore, in this case, a releasable linker is not required. For other targets that are in the cytosol rather than the endosome, such as SHP1 / 2, TC-PTP, DGK, TGFβ, and STING, the release of the free drug from the target-payload conjugate is required to escape the endosome. Taken together, this covert pathway system provides a versatile platform for the specific in vivo delivery of a large number of payloads to exhausted CAR T cells.

[0047] These and other features, aspects and advantages of the present invention will be better understood from the following examples.

[0048] method: Anti-CD19 CAR T cell depletion and drug treatment: Anti-CD19 CAR T cells were co-cultured with Raji at a 1:1 ratio in 6-well plates, with fresh Raji cells added to the same wells every 12 hours. Killing activity and co-inhibitory markers were quantified by flow cytometry counting. For drug treatment, after four stimulations with Raji cells, exhausted anti-CD19 CAR T were further incubated with different concentrations of drugs for 12 hours and then quantified in the same way.

[0049] Targeted Ligand Binding Assays Fusion receptor-positive cells were incubated with various concentrations of a specific ligand-dye molecule for 30 min at 4° C. After incubation, cells were washed twice with PBS and then submitted to flow cytometry. MFI or percent shift was used for binding curves and Kd calculations.

[0050] material Cell lines and human T cells MDAMB-231 cells and MDA-MB-231 CD19 + DMEM (Gibco) containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin was used to culture the cells. Peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood obtained from healthy volunteers by Ficoll density gradient centrifugation (GE Healthcare Lifesciences, #17-5442-02). Pure CD3+ T cells were enriched from PBMCs using EasySep™ Human T Cell Isolation Kit (STEM CELL technologies, #17951).

[0051] Evaluation of potential payloads to rejuvenate exhausted CAR T cells in vitro Anti-CD19 CAR T cells were cultured in 12-well plates to incubate with CD19 + 2x10 cells per well in a 1:1 ratio with Raji cells 6 CAR T and 2x10 6 The cells were co-incubated at a density of 10 Raji cells per well. Fresh Raji cells were added every 12 hours for three times, and then the Raji cell population, lytic efficacy, and co-inhibitory receptors were tested to confirm CAR T cell exhaustion. The lytic efficacy was quantified using flow cytometry and luciferase-based assays. This cell mixture was then plated in 96-well plates at approximately 2x10 cells per well to test the rejuvenation potency of potential payloads. 5 100 cells were transferred and different concentrations of drugs were added. After 12 hours, the Raji cell population, lytic effects, and co-inhibitory receptors were re-examined and compared to the PBS-treated group. EXAMPLES

[0052] Example 1. In vitro depletion of anti-CD19 CAR T cells In this example, we demonstrated a model of exhausted CAR T cells. Briefly, Fig. 1a shows that Raji cells, a type of B lymphoma cells, were co-cultured with FMC63 CAR T cells, a type of anti-CD19 CAR T cells, as described in the method section, and fresh Raji cells were added to the co-culture every 12 hours for three consecutive days. Fig. 1b shows that after three stimulations with fresh Raji cells in vitro, these CAR T cells became exhausted as indicated by a reduced killing effect. Co-cultured K562 cells served as a negative control.

[0053] Example 2. Designing a secret path In this example, we show a diagram of the secret pathway of delivery payload drugs to targeted cell types. FKBP or anti-FITC is linked to FRa as a fusion receptor. The fusion receptor can bind to targeting ligands FK506 or FITC linked to a specific drug payload. Due to the nature of FRa, the fusion receptor continuously internalizes and delivers the payload linked to FK506 or FITC into CAR T cells. Thus, when CAR T binds to a target cell via CAR, in this example, the anti-CD19 molecule on the surface of CAR T binds to CD19 on the cancer cell, and the delivery payload drug in CAR T can exert its function, i.e., regulate CAR T activity based on the payload function. For example, some payload drugs can be directed to act on PD-1, CTLA4, or LAG3 T cell function regulating molecules to rejuvenate CAR T as needed.

[0054] Example 3. Reversal of exhausted anti-CD19 CAR T cells by TLR7 agonists In this example, we show that the targeting ligands FK506 or FITC successfully bind to the respective fusion receptors (FKBP or FITC-AF647 linked to the folate receptor by G4S). In this example, the payload drug is the imaging agent rhodamine to show the payload distribution in Jurkat cells transfected with the fusion receptor. The binding affinity of FK506 and FITC was calculated by competitive binding. FK506-rhodamine shows Kd=3.39nM to the FKBP-FR fusion receptor, while FITC-AF647 shows Kd=8.03nM to the anti-FITC-FR.

[0055] Example 4. Design of potential releasable and non-releasable FK506-TLR7 and FITC-TLR7 agonists In this example, we provided targeting ligand FK506 conjugated with Toll-like receptor 7 agonist with the following structure to treat exhausted CAR T cells. When exhausted CAR T cells are targeted with TLR7 agonist payload drug, a dose-dependent pattern of increased killing is observed. Thus, IFNγ expression level, an indicator of CAR T activity, increased dose-dependently compared to payload drug concentration. Expression level of Tim3, an inhibitory molecule of T cell effect, showed the opposite dose-dependence. That is, Tim3 expression decreased with increasing payload drug concentration.

[0056] The FK506-TLR7 agonist conjugate has the structure shown below: It should have one of these: TIFF0007623935000033.tif51128.

[0057] Similarly, the fluorescein-TLR7 agonist conjugate should have one of the structures shown below and will rejuvenate exhausted CAR Ts if the functional CAR T has an anti-FITC fusion receptor. TIFF0007623935000034.tif48128

[0058] Example 5. Design of other potential payloads to rejuvenate exhausted CAR T cells In this example, we provide a list of other potential payload structures that may reverse CAR T attrition with potency in the low nanomolar range.

[0059] 1) A TC-PTP phosphatase inhibitor should have the following structure: TIFF0007623935000035.tif24144

[0060] The above mentioned phosphatase inhibitors can be linked to fluorescein or FK506 (tacrolimus) as follows. TIFF0007623935000036.tif79128

[0061] 2) STING agonists should have the following structure: TIFF0007623935000037.tif51128

[0062] Example 6. Design of a spacer between the targeting ligand and the potential payload Below is a list of spacers that can be used to link the targeting ligand and any potential payload. TIFF0007623935000038.tif58128

[0063] Example 7. Evaluation of the ability of phosphatase inhibitors and TLR7 agonists to rejuvenate exhausted CAR T cells One of the major limitations of CAR T cell therapy in solid tumors is their tendency to become exhausted after repeated stimulation with cancer antigens. However, this phenomenon is not unique to CAR T cells and may be related to chronic viral infections. 4 and tumor-infiltrating lymphocytes 5 The reversibility of the T cell exhaustion phenotype has been demonstrated in a study in which T cells isolated from solid tumor tissues showed high levels of INFγ secretion and killing activity when restimulated after an overnight withdrawal from antigen ("rest"). 57 However, in a more clinically relevant manner, it would be attractive to rejuvenate T cells using commercially available therapeutics that block inhibitory signaling or activate T cells through other pathways. Antibodies targeting checkpoint inhibitors (i.e., PD-1, CTLA-4, etc.) have shown some success in the clinic and in solid tumors. 151 However, it is often found that a combination of two or more targets is necessary. In addition, antibody therapy has the disadvantage of poor penetration into solid tumors. This may have led to the paucity of reports of combination therapy of CAR T cells and checkpoint blockade (ICB) in solid tumors.

[0064] Inhibition of phosphatases such as SHP1 / 2 and TC-PTP, which mediate TCR deactivation, is a potential approach to block sustained CAR T signaling. SHP1 / 2 phosphatases are responsible for mediating signals from PD-1 and other exhaustion markers. Data show that SHP1 / 2 phosphatase inhibitors or silencing can increase the activity of T cells and CAR T cells 6-9 TC-PTP has been shown to be a critical player in T cell activation signaling. Mice with T cell-specific TC-PTP deficiency are more susceptible to inflammation and autoimmunity due to increased antigen-induced T cell activation. TC-PTP inactivates Src family kinases downstream of the TCR, thereby contributing to the threshold for TCR activation. 11 Although knockout experiments and small molecule inhibitors of these phosphatases have shown potent effects on lowering the TCR threshold and enhancing T cell activity, none of them have been used in CAR T therapy. Representative SHP1 / 2 inhibitors are TIFF0007623935000039.tif has a structure of 46128.

[0065] Another approach to rejuvenate T cells is to enhance their activity by binding to antigen-independent innate immune receptors. It is known that certain pathogen pattern recognition (PPR) receptors, including toll-like receptors (TLRs), are expressed on non-myeloid cell populations, including T cells, and can be activated in the same way. Studies have also shown that costimulating TCR signaling with TLR7 / 8 agonists activates CD8 T cells and increases INFγ secretion. 13However, none of these agonists have been used in CAR T therapy to reactivate T cells or to change the immunosuppressive microenvironment due to the strong side effects of systemic administration of TLR agonists. The use of TLR agonists for cancer immunotherapy is also hindered by the controversial action of TLR agonists on tumor cells. Therefore, targeted delivery of potential payloads to CAR T cells is highly desirable. A potent TLR7 agonist has been discovered in the literature (Figure 7A), which is about 40 times more potent than FDA-approved imiquimod.

[0066] To assemble an in vitro screening model, anti-CD19 CAR T cells were exposed to four rounds of CD19-positive Raji cell addition, as shown in Figure 6. Anti-CD19 CAR T cells entered an exhausted state, as indicated by a gradual decrease in lytic activity and an increase in co-inhibitory markers in the in vitro co-culture model. It is noteworthy that the culture medium is critical for the induction of CAR T exhaustion and should be kept the same without fresh replenishment or replacement during the whole process. This suggests that soluble components released into the medium by cancer cells and / or CAR T cells, most likely immunosuppressive cytokines and modulators (such as adenosine), play a central role in this process. This also suggests that the exhaustion of CAR T cells generated by this in vitro model is not irreversible but is in an adaptive state.

[0067] Preferred TLR7 agonists and (highly homologous to TC-PTP) 26 )PTP1b inhibitors 27It was shown that treatment of PTP1b with already exhausted CAR T cells could reactivate CAR T cells compared to the untreated group (Figure 7). However, no significant changes were observed in the expression levels of co-inhibitory markers, except Tim3. As shown in Figure 7C, PTP1b inhibitors generally do not show as strong reactivating effects as TLR7 agonists. Without being bound by theory, this result may be due to the current in vitro screening model, in which the "reversion" of exhaustion is studied rather than the "prevention" of exhaustion, and the phosphatase is already "silenced" in the exhausted state, and therefore its inhibition has no or little effect. An improved screening model for future studies will test the effects of phosphatase inhibitors and other drugs, focusing on the "prevention" of exhaustion by adding the drugs at the beginning of the preculture and keeping the rest of the setup the same. Thus, it may be possible to see whether phosphatase inhibition can attenuate the tonic signaling of CAR T while retaining functional killing activity. We will primarily focus on TLR7 agonists for the following studies.

[0068] Because TLR7 is one of the four TLR family members that reside in endosomes, a non-releasable linker between TLR7 agonists and our secret pathway targeting ligands is predicted to preserve their TLR7 agonist function. 28To achieve this, several TLR7 agonist analogs were prepared and tested to find a suitable derivatization site for conjugation. As shown in Figure 8, TLR7 agonists with a CH2OH extension on the piperidine ring have significantly higher activity compared to the parent drug. Therefore, this derivatization site is used for non-releasable conjugates. A self-immolative form linked by a disulfide bond was also synthesized. To understand the distance required for this TLR7 agonist to reach its own target, three different lengths of linkers (PEG3, 6, 16) were created between FITC and the TLR7 agonist for non-releasable FITC-TLR7. As shown in Figure 9C, all of these non-releasable forms were effective to some extent, but the PEG6 compound showed the best dose-dependent response. These results indicate that the TLR7 agonist can dock with TLR7 by reaching while bound to FITC-FR or by jumping between the TLR and FITC-FR. Under these conditions, the length of the intermediate linker is not a determining factor (Figure 9D-E). Since the non-releasable FITC-TLR7 is trapped inside endosomes and the volume of each endosome is much smaller than the cytosol, intraendosomal TLR7 can reach functional concentrations very quickly and rapidly, resulting in an IC 50 becomes smaller.

[0069] Example 8. Other potential payloads to reverse / prevent CAR T cell exhaustion Besides TLR7 agonists, there are several other potential payloads that may reverse / prevent CAR T cell exhaustion, as described below. Some of the targets are currently IC suitable for our targeted drug delivery approach. 50 Although there may be no agonists or inhibitors with similar properties, they are still worth noting and may be explored by other mechanisms of inhibition, such as CRISPR or targeted microRNA delivery approaches.

[0070] STING agonists Simulator of IFN Genes (STING) is a master adaptor involved in cytosolic DNA sensing and following IFN-β production. STING binds weakly to sdDNA but strongly to the endogenous cyclic dinucleotide GMP-AMP (cGAMP) synthesized by cGMP-AMP synthase (sGAS). It is mainly expressed in macrophages, T cells, various DCs, endothelial cells, and selected fibroblasts and epithelial cells. Studies of STING have mainly focused on its function in macrophages and dendritic cells, and recently, several groups have observed a direct effect of STING activation in T cells. 40 STING agonists may have similar pro-inflammatory effects on T cells. ADU-S100 is one of many STING agonists that have been explored in the clinic.

[0071] DGK-α inhibitors Diacylglycerol kinase-α (DGK-α), together with IP3, converts diacylglycerol (DAG), a second messenger of TCR signaling, to phosphatidic acid (PA). DGK is more highly expressed in CD8 TILs than in CD8-NILs, and inhibition of DGK promotes ERK phosphorylation and lytic degranulation. 41 It also restores the lytic function of CAR TILs isolated from in vivo 5 Some DGK inhibitor structures are as follows: TIFF0007623935000040.tif54145

[0072] TGFβRI (ALK5) inhibitor TGFβ is known to have immunosuppressive functions in many immune cells, including T cells, B cells, and macrophages. Blockade of the TGFβ type I receptor (TGFβRI, also known as ALK5) in T cells reverses the immunosuppressive environment of tumors. 42Small molecule inhibitors are being explored, with galunisertib (LY2157299 monohydrate) and EW-7197 being tested in the clinic. 43-44 .

[0073] The TGFβ inhibitor structure is as follows: TIFF0007623935000041.tif47128

[0074] EZH2 inhibitors Enhancer of Zeste Homolog 2 (EZH2) is a histone H3K27 methyltransferase that strongly correlates with Treg function. Genetic or pharmacological disruption of EZH2 conferred proinflammatory function to tumor-infiltrating Tregs. 45 Exhausted CTLs in chronic viral infections are also characterized by unique epigenetic changes. 174 ,EZH2 inhibitors may be able to reverse this wasting state.,Several small molecule EZH2 inhibitors have been developed, including CPI1205, EPZ6438, and GSK126.

[0075] Thus, the FITC-FR fusion receptor and the corresponding FITC-targeted immune agonist payload provide a universal platform to monitor and control the homing and persistence of CAR T cells in solid tumors. As the FITC-FR fusion receptor is expressed independently of the CAR construct, this approach can be easily adapted to CAR T cells against any antigen. The modular design of the targeting ligand-payload conjugate also allows for easy switching and improvement. This approach combines the benefits of cell therapy and small molecule-based targeted drug delivery, which may require extra characterization of both the engineered cells and the corresponding ligand.

[0076] The success of CAR T cells in solid tumors will almost certainly require the combination of multiple approaches targeting other players in the microenvironment, such as the degradation of extracellular matrix by PI3K kinase inhibitors, reprogramming anti-inflammatory M2 macrophages to a pro-inflammatory M1 phenotype, and upregulation of reduced MHC molecule levels on cancer cells. Thus, more and more combination therapy trials are being conducted in preclinical and clinical settings. However, at the same time, careful testing and management of the CAR T cells themselves is essential and they must first be optimized in preclinical studies using simple but robust systems such as FITC-FR fusion receptors before reaching humans.

[0077] References: TIFF0007623935000042.tif184165TIFF0007623935000043.tif212165TIFF0007623935000044.tif220166TIFF0007623935 000045.tif223166TIFF0007623935000046.tif222166TIFF0007623935000047.tif222166TIFF0007623935000048.tif33166

Claims

1. At least two of the following ingredients: a first component that is a conjugate comprising a targeting ligand covalently linked to a payload drug; and A second component which is a fusion receptor comprising a target ligand binding module linked to a membrane anchor module. A system for rejuvenating exhausted chimeric antigen receptor (CAR) T cells, comprising: The fusion receptor is expressed on the surface of the CAR T cell, the payload drug is a Toll-like receptor 7 (TLR7) agonist; the target ligand and the target ligand binding module are fluorescein isothiocyanate (FITC) and anti-FITC scFv, respectively, or tacrolimus (FK506) and FK506 binding protein (FKBP), respectively; The membrane anchor module is a folate receptor, the target ligand binding module of the second component recognizes the target ligand in the first component with high affinity to form a complex; Payload drugs reactivate exhausted CAR T cells via an antigen-independent pathway, The membrane anchor module mediates internalization of the two-component complex into exhausted CAR T cells. The system.

2. The system of claim 1, wherein the membrane anchor module is folate receptor alpha (FRα).

3. 3. The system of claim 1 or 2, wherein the first component comprises a releasable linker between the targeting ligand and the payload drug.

4. 3. The system of claim 1 or 2, wherein the first component comprises a non-releasable linker between the targeting ligand and the payload drug.

5. The system of any one of claims 1 to 4, wherein the binding affinity between the target ligand and the ligand binding module is in the sub-nM range.

6. TLR7 agonists, The system according to any one of claims 1 to 5, having a structure as follows:

7. The targeting ligand and the targeting ligand binding module are fluorescein isothiocyanate (FITC) and an anti-FITC scFv, respectively, and the first component comprises: The system of any one of claims 1 to 6, wherein the fluorescein isothiocyanate-TLR7 agonist has the structure:

8. The target ligand and the target ligand binding module are tacrolimus (FK506) and FK506 binding protein (FKBP), respectively, and the first component is The system according to any one of claims 1 to 6, wherein the FK506-TLR7 agonist has the structure:

9. The method of claim 8, wherein the targeting ligand and the targeting ligand binding module are fluorescein isothiocyanate (FITC) and anti-FITC scFv, respectively, and the first component comprises the following: The system according to any one of claims 1 to 6, wherein the system is one of:

10. The first component has, between the targeting ligand and the payload drug, the following structure: The system of any one of claims 1 to 6, comprising a spacer selected from the group consisting of:

11. A conjugate comprising a targeting ligand covalently linked to a payload drug via a releasable or non-releasable linker.

1. A pharmaceutical composition for use in a method for rejuvenating exhausted CAR T cells, comprising: the exhausted CAR T cells express a fusion receptor comprising a target ligand binding module and a membrane-bound receptor module; the payload drug is a TLR7 agonist; the target ligand and the target ligand binding module are FITC and anti-FITC scFv, respectively, or FK506 and FKBP, respectively; the membrane-bound receptor module is a folate receptor; The target ligand binding module binds to the target ligand to form a complex; The membrane-bound receptor module mediates internalization of the complex into the CAR T cell. Payload drugs reactivate CAR T cells through an antigen-independent pathway The pharmaceutical composition.

12. A CAR T cell expressing a fusion receptor, the fusion receptor comprising a target ligand binding module and a membrane-bound receptor module.

1. A pharmaceutical composition for use in a method for rejuvenating exhausted CAR T cells, comprising: The pharmaceutical composition comprises: A conjugate comprising a targeting ligand covalently linked to a payload drug via a releasable or non-releasable linker. It is used in combination with the conjugate is administered following depletion of CAR T cells expressing the fusion receptor; the payload drug is a TLR7 agonist; the target ligand and the target ligand binding module are FITC and anti-FITC scFv, respectively, or FK506 and FKBP, respectively; the membrane-bound receptor module is a folate receptor; The target ligand binding module binds to the target ligand to form a complex; The membrane-bound receptor module mediates internalization of the complex into the CAR T cell. Payload drugs reactivate CAR T cells through an antigen-independent pathway The pharmaceutical composition.

13. A construct comprising a CAR and a fusion receptor, the fusion receptor comprising a target ligand binding module and a membrane-bound receptor module.

1. A pharmaceutical composition for use in a method for rejuvenating exhausted CAR T cells, comprising: The method comprises: transfecting T cells with the construct to generate CAR T cells expressing the CAR and the fusion receptor; After exhaustion of the CAR T cells, A conjugate comprising a targeting ligand covalently linked to a payload drug via a releasable or non-releasable linker. and contacting the Including, the payload drug is a TLR7 agonist; the target ligand and the target ligand binding module are FITC and anti-FITC scFv, respectively, or FK506 and FKBP, respectively; the membrane-bound receptor module is a folate receptor; The target ligand binding module binds to the target ligand to form a complex; The membrane-bound receptor module mediates internalization of the complex into the CAR T cell. The payload drug reactivates the CAR T cells via an antigen-independent pathway, thereby rejuvenating the CAR T cells. The pharmaceutical composition.

14. 14. The pharmaceutical composition of any one of claims 11 to 13, wherein the payload drug performs its function in the endosomes of exhausted CAR T cells, and the targeting ligand and the payload drug are linked by a non-releasable linker.

15. 14. The pharmaceutical composition of any one of claims 11 to 13, wherein the payload drug performs its function as a free drug in the cytosol of the exhausted CAR T cells, and the targeting ligand and the payload drug are linked by a releasable linker.

16. The pharmaceutical composition of any one of claims 11 to 15, wherein the membrane-bound receptor module is folate receptor alpha (FRa).

17. TLR7 agonists, 17. The pharmaceutical composition of any one of claims 11 to 16, having the structure:

18. The method of claim 17, wherein the targeting ligand and the targeting ligand binding module are fluorescein isothiocyanate (FITC) and an anti-FITC scFv, respectively, and the conjugate comprises The pharmaceutical composition of any one of claims 11 to 17, which is a fluorescein isothiocyanate-TLR7 agonist having the structure:

19. The method of claim 1, wherein the target ligand and the target ligand binding module are tacrolimus (FK506) and FK506 binding protein (FKBP), respectively, and the conjugate comprises: The pharmaceutical composition of any one of claims 11 to 17, which is an FK506-TLR7 agonist having the structure:

20. The method of claim 1, wherein the targeting ligand and the targeting ligand binding module are fluorescein isothiocyanate (FITC) and anti-FITC scFv, respectively, and the conjugate comprises the following: The pharmaceutical composition according to any one of claims 11 to 17, wherein the pharmaceutical composition is one of:

21. The conjugate has the following structure between the targeting ligand and the payload drug:

18. The pharmaceutical composition of any one of claims 11 to 17, comprising a spacer selected from the group consisting of:

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