US-12622973-B2 - Rejuvenation of CAR T cell

Abstract

A payload of drug conjugated to a targeting ligand specifically designed to deliver to exhausted CAR T cells to rejuvenate these CAR T cells is provided herein. The targeted CAR T cells are modified with a fusion receptor which can bind to the targeting ligand and internalize the conjugated payload of drug to execute its regulatory function to exhausted CAR T cell.

Inventors

• Philip Stewart Low
• Boning Zhang
• John V. Napoleon

Assignees

• PURDUE RESEARCH FOUNDATION

Dates

Publication Date

20260512

Application Date

20231221

Claims (18)

1. 1 . A system to rejuvenate an exhausted classical CAR T cell comprising at least two components: a first component that is a conjugate comprising a targeting ligand covalently linked to a payload drug; and a second component that is a targeting ligand binding module linked to a membrane-anchoring module, wherein: the payload drug is a Toll Like Receptor 7 (TLR7) agonist, the targeting ligand and the targeting ligand binding molecule are fluorescein isothiocyanate (FITC) and an anti-FITC antibody or fragment thereof, respectively; or tacrolimus (FK506) and FK506-binding protein (FKBP), respectively, the membrane-anchoring module is a folate receptor, the targeting ligand binding module of the second component recognizes the targeting ligand in the first component with high affinity to form a complex, the payload drug re-activates the CAR T cell through an antigen-independent pathway, and the membrane-anchoring module mediates internalization of the at least two components complex into the exhausted classical CAR T cell.
2. 2 . The system according to claim 1 , wherein the membrane-anchoring module is a folate receptor alpha (FRα).
3. 3 . The system according to claim 1 , wherein the first component comprises a releasable linker between the targeting ligand and the payload drug.
4. 4 . The system according to claim 1 , wherein the first component comprises a non-releasable linker between the targeting ligand and the payload drug.
5. 5 . The system according to claim 1 , wherein the binding affinity between the targeting ligand and the ligand-binding module is in a sub-nanomolar range.
6. 6 . The system according to claim 1 , wherein the TLR7 agonist has the structure of
7. 7 . The system according to claim 1 , wherein targeting ligand and the targeting ligand binding molecule are fluorescein isothiocyanate (FITC) and an anti-FITC antibody or fragment thereof, respectively, and the first component is a fluorescein isothiocyanate-TLR7 agonist having the structure:
8. 8 . The system according to claim 1 , wherein the targeting ligand and the targeting ligand binding molecule are tacrolimus (FK506) and FK506-binding protein (FKBP), respectively, and the first component is a FK506-TLR7 agonist having the structure:
9. 9 . The system according to claim 1 , wherein the targeting ligand and the targeting ligand binding molecule are fluorescein isothiocyanate (FITC) and an anti-FITC scFv, respectively, and wherein the first component is one of the following:
10. 10 . The system according to claim 1 , wherein the first component comprises a spacer between the targeting ligand and the payload drug, wherein the spacer is selected from the group consisting of the following structures: wherein n is 1-12.
11. 11 . A method to rejuvenate an exhausted CAR T cell, comprising: a. providing to the exhausted CAR T cell a first component comprising a conjugate comprising, a targeting ligand covalently linked to a payload of drug through a releasable linker or a non-releasable linker; b. providing said exhausted CAR T cell a second component comprising a fusion receptor, wherein the fusion receptor comprises a targeting ligand binding module linked to a membrane-anchoring module; c. wherein the targeting ligand binding module of the second component binds to the targeting ligand in the first component to form a complex, d. the membrane-bound receptor module mediates internalization of the complex into the exhausted CAR T cell; and e. the payload drug re-activates the CAR T cell through an antigen-independent pathway; wherein: the payload drug is a Toll Like Receptor 7 (TLR7) agonist, the targeting ligand and the targeting ligand binding molecule are fluorescein isothiocyanate (FITC) and an anti-FITC antibody or fragment thereof, respectively, or tacrolimus (FK506) and FK506-binding protein (FKBP), respectively, and the membrane-anchoring module is a folate receptor.
12. 12 . The method according to claim 11 , wherein the payload drug executes its function within the endosome of the exhausted CAR T cell, and the targeting ligand and the payload drug are linked by a non-releasable linker.
13. 13 . The method according to claim 11 , wherein the payload drug executes its function as a free drug in the cytosol of the exhausted CAR T cell, and the targeting ligand and the payload drug are linked by a releasable linker.
14. 14 . The method according to claim 11 , wherein the TLR7 agonist has the structure:
15. 15 . The method according to claim 11 , wherein the first component is a targeting ligand and the targeting ligand binding molecule are fluorescein isothiocyanate (FITC) and an anti-FITC antibody or fragment thereof, respectively, and the first component is a fluorescein isothiocyanate-TLR7 agonist having the structure:
16. 16 . The method according to claim 11 , wherein the targeting ligand and the targeting ligand binding molecule are tacrolimus (FK506) and FK506-binding protein (FKBP), respectively, and the first component is a FK506-TLR7 agonist having the structure:
17. 17 . The method according to claim 11 , wherein the targeting ligand and the targeting ligand binding molecule are fluorescein isothiocyanate (FITC) and an anti-FITC scFv, respectively, and wherein the first component is one of the following;
18. 18 . The method according to claim 11 , wherein the linker between the targeting ligand and the payload drug is selected from the group consisting of the following structures: wherein n is 1-12.

Description

RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 17/266,509, filed on 5 Feb. 2021, which is a U.S. national stage filing under 35 U.S.C. 371 from International Application No. PCT/US2019/042726, filed on 21 Jul. 2019, and published as WO 2020/033129 on 13 Feb. 2020, which claims the benefit of U.S. Provisional Application Ser. No. 62/715,666, filed 7 Aug. 2018, the contents of which are specifically incorporated herein by reference in their entirety. FIELD OF INVENTION This disclosure provides a system to rejuvenate cancer antigen exhausted chimeric antigen receptor T (CAR T) cells. Specifically, the system comprising a fusion receptor in a classical CAR construct, wherein the fusion receptor provides a ligand binding module that recognizes a high affinity ligand-payload drug conjugate to deliver payload of drugs that are designed to either block the inhibitory signaling in the exhausted CAR T, or to re-activate CAR T through an antigen independent pathway. BACKGROUND The field of chimeric antigen receptor (CAR) T cell therapy has made tremendous progress over the last two decades. The CAR construct consists of four parts: (1) an extracellular binding moiety against tumor specific antigen, (2) a hinge domain, (3) a transmembrane domain and (4) a combination of various activation domains, for example, CD28, 4-1BB and CD3ζ chain. The most impressive successes have been seen in CAR T therapy against CD19 positive B cell leukemias, where more than 80% complete remission rate has been achieved in several clinical trials. In contrast, the treatment of solid tumor by CAR T has so far proven to be more challenging. Great successes have been achieved in preclinical study using syngeneic mice model or xenograft tumor model in immunodeficient mice. However, none of the clinical trials involving solid tumor, for example, breast cancer, ovarian cancer or lung cancer has shown improvement compare to control. In general, it suffers from the same limitations that are faced by other adoptive cell therapies, including (1) poor tumor penetration; (2) hypoxic pressure; (3) are immunosuppressive tumor microenviroment which includes tumor associated macrophages, fibroblasts and suppressive cytokines1. CAR T cells, as same as tumor infiltrated lymphocytes (TIL), can be re-educated by this suppressive microenviroment and turn to a “hypofunctional” status, which is characterized by overexpression of co-inhibitory molecules (i.e., PD-1, Tim-3, LAG-3 etc.) decreased INFγ secretion and killing capability. Data from the patient samples of ovarian cancer shows that the majority of PD-1+CD8+ T cell lacked expression of CD127 which is known to be important for the effector-to-memory transition in T cell. Overexpression of LAG-3 also negatively correlated with the effector function of TCR specific CD8+ TIL. In addition, PD-1+ LAG-3+ double positive T cell exhibited lower INFγ production2. Similarly, NY-ESO-1 TCR specific human T cell became hypofunctional in mice solid tumor model, showing high expression of the co-inhibitory molecules and less efficient anti-tumor effect, due to both the microenvironment and the constant activation of T cell by the continuous exposure to antigen3. Therefore, a reversion of the suppressive microenviroment, more importantly, a rejuvenation of the exhausted CAR T cell is highly desired for a better solid tumor treatment. SUMMARY OF THE INVENTION This disclosure provides system to rejuvenate an exhausted classical CAR T cell. The system comprises at least two components: a first component is a conjugate comprising a targeting ligand covalently linked to a payload of drug; and a second component is a targeting ligand binding module linked to membrane-anchoring 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 either blocks the inhibitory signaling of the exhausted CAR T, or re-activates said CAR T through an antigen independent pathway. The membrane-anchoring module mediates internalization of the two component complex into the exhausted CAR T cell. In some preferred embodiment, the aforementioned targeting ligand of the first component is folate, FITC or FK506. In some preferred embodiment, the aforementioned targeting ligand binding module of the second component comprises a folate receptor, an anti-FITC antibody fragment or FKBP. In some preferred embodiment, the aforementioned membrane-anchoring module is a folate receptor. In some preferred embodiment, the aforementioned first component comprises a releasable linker between the targeting ligand and the payload drug. In some preferred embodiment, the aforementioned first component comprises a non-releasable linker between the targeting ligand and the payload drug. In some preferred embodiment, the binding affinity between aforementioned targeting ligand and the targeting ligand-binding modul