WO2025085511A1

WO2025085511A1 - Systems and methods for polymer-mediated activation of engineered cells

Info

Publication number: WO2025085511A1
Application number: PCT/US2024/051567
Authority: WO
Inventors: Suzie Hwang Pun; Clinton HEINZE; Trey PICHON; Joshua Gustafson; Michael C. Jensen
Original assignee: University of Washington; Seattle Childrens Hospital
Current assignee: University of Washington; Seattle Childrens Hospital
Priority date: 2023-10-18
Filing date: 2024-10-16
Publication date: 2025-04-24
Anticipated expiration: 2026-04-18

Abstract

Disclosed are systems for polymer-mediated activation of an engineered cell. The disclosed systems include (a) a polymer displaying a universal epitope and a moiety that specifically binds to a component of a tissue or cell of interest and (b) an engineered cell comprising (i) an engineered receptor that specifically recognizes the universal epitope via an extracellular binding domain and (ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter driven by an activation signal from an intracellular signaling domain of the engineered receptor upon engagement of the receptor with the universal epitope. Also disclosed are methods for delivering a therapeutic protein to the site of a tissue or cell of interest by administering the disclosed polymer and engineered cell. In some variations, the engineered cell is a chimeric antigen receptor (CAR) cell and/or the moiety binds to an extracellular matrix component. In some aspects, the system is useful in methods for treating a solid tumor cancer.

Description

SYSTEMS AND METHODS FOR POLYMER-MEDIATED ACTIVATION OF ENGINEERED CELLS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Application No. 63/591,378, filed on October 18, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Copy, created on September 20, 2024, is named “3195-P1322WO- UW_Seq_Listing_ST26.xml” and is 41,704 bytes in size.

BACKGROUND

[0003] Chimeric antigen receptor (CAR) T cell therapy has demonstrated unprecedented efficacy against hematological malignancies, producing high remission rates and resulting in six FDA-approved therapies to date. See Maude et al., N. Engl. J. Med. 371 : 1507-1517, 2014; Brentjens et al., Sci. Transl. Med. 5: 177ra38, 2013; Lee et al., Lancet 385:517-528, 2015. Despite this clinical success, currently available CARs employ a relatively simple design. All FDA-approved products contain a single monovalent CAR with unregulated function post-infusion into patients. The ramifications of this range from hypofunctional behavior to toxicity-inducing over-reactivity. Tumor heterogeneity, in the form of antigen diversity and tumor plasticity, and subsequent antigen loss are primary factors that lead to CAR T cell underperformance. See Majzner & Mackall, Cancer Discov. 8:1219-1226, 2018. CARs specific for a single target are unable to handle tumors with diverse antigen profiles, resulting in an outgrowth of tumor cells unrecognizable to the CAR T cells. On the flip side, current CAR T cells lack specificity, driving systemic toxicity issues, such as cytokine release syndrome (CRS) (see Bonifant et al. , Mol. Ther. Oncolytics 3 : 16011 , 2016), or site-specific damage due to on-target, off-tumor reactivity (see Morgan et al., Mol. Ther. 18:843-851, 2010; Parkhurst et al., Mol. Ther. 19:620-626, 2011). To deal with these deficiencies, next generation CAR T cell systems should have the ability for multivalent targeting, enhanced control over cell activity, and selective activation within tumors.

SUMMARY

[0004] In one aspect, the present disclosure provides a system for polymer-mediated activation of an engineered cell. The system generally includes (a) a polymer comprising a plurality of repeating units forming a polymer backbone, wherein the polymer comprises (i) a universal epitope, and (ii) a moiety that specifically binds to a component of a tissue or cell of interest; and (b) an engineered cell comprising (i) an engineered receptor that specifically recognizes the universal epitope, wherein the engineered receptor is constitutively expressed by the engineered cell, and wherein the engineered receptor comprises (A) an extracellular binding domain that specifically binds to the universal epitope, and (B) an intracellular signaling domain derived from a cell surface receptor, and (ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the intracellular signaling domain upon engagement of the extracellular binding domain with the universal epitope.

[0005] In particular embodiments of a system as above, the engineered cell is a CAR immune cell (e.g., a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell) and the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment. Accordingly, in some such variations, the system is a system for polymer-mediated activation of a CAR immune cell generally including (a) a polymer comprising a plurality of repeating units forming a polymer backbone, wherein the polymer comprises (i) a universal chimeric antigen receptor (CAR) epitope, and (ii) a moiety that specifically binds to an extracellular matrix (ECM) component of a tumor microenvironment; and (b) a CAR immune cell comprising (i) a CAR that specifically recognizes the universal CAR epitope, wherein the CAR is constitutively expressed by the CAR immune cell, and (ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the CAR upon engagement of CAR with the universal CAR epitope.

[0006] In another aspect, the present disclosure provides a method for expressing a therapeutic protein at the site of a tissue or cell of interest in a subject using a system as described herein. The method generally includes administering to a subject an effective regimen of (a) a polymer comprising a plurality of repeating units forming a polymer backbone, wherein the polymer comprises (i) a universal epitope, and (ii) a moiety that specifically binds to a component of a tissue or cell of interest; and (b) an engineered cell comprising (i) an engineered receptor that specifically recognizes the universal epitope, wherein the engineered receptor is constitutively expressed by the engineered cell, and wherein the engineered receptor comprises (A) an extracellular binding domain that specifically binds to the universal epitope, and (B) an intracellular signaling domain derived from a cell surface receptor, and (ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the intracellular signaling domain upon engagement of the extracellular binding domain with the universal epitope; wherein the polymer specifically binds to the component of the tissue or cell of interest in the subject, thereby localizing the polymer to the site of the tissue or cell, and wherein the engineered receptor specifically binds to the universal epitope of the localized polymer, thereby inducing expression of the therapeutic protein at the site of the tissue or cell of interest.

[0007] In another aspect, the present disclosure provides a method for treating a solid tumor cancer using a system as described herein. The method generally includes administering to a subject having the solid tumor cancer an effective regimen of (a) a polymer comprising a plurality of repeating units forming a polymer backbone, wherein the polymer comprises (i) a universal chimeric antigen receptor (CAR) epitope, and (ii) a moiety that specifically binds to an extracellular matrix (ECM) component of the tumor microenvironment; and (b) a CAR immune cell comprising (i) a CAR that specifically recognizes the universal CAR epitope, wherein the CAR is constitutively expressed by the CAR immune cell, and (ii) a transgene comprising a polynucleotide encoding an anticancer therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the CAR upon engagement of CAR with the universal CAR epitope.

[0008] Representative embodiments of these aspects are further set forth below. Embodiments

[0009] Embodiment 1. A system for polymer-mediated activation of an engineered cell, the system comprising:

(a) a polymer comprising a plurality of repeating units forming a polymer backbone, wherein the polymer comprises

(i) a universal epitope, and

(ii) a moiety that specifically binds to a component of a tissue or cell of interest; and

(b) an engineered cell comprising

(i) an engineered receptor that specifically recognizes the universal epitope, wherein the engineered receptor is constitutively expressed by the engineered cell, and wherein the engineered receptor comprises

(A) an extracellular binding domain that specifically binds to the universal epitope, and

(B) an intracellular signaling domain derived from a cell surface receptor, and

(ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the intracellular signaling domain upon engagement of the extracellular binding domain with the universal epitope.

[0010] Embodiment 2. The system of Embodiment 1, wherein the universal epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope.

[0011] Embodiment 3. The system of Embodiment 1 or 2, wherein the universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tissue or cell of interest. [0012] Embodiment 4. The system of Embodiment 3, wherein the mask comprises a self-immolative linker.

[0013] Embodiment 5. The system of Embodiment 4, wherein the self-immolative linker comprises a difluorophenyl ester or a p-aminobenzylalchohol.

[0014] Embodiment 6. The system of Embodiment 3, wherein the universal epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

[0015] Embodiment 7. The system of any one of Embodiments 1 to 6, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide.

[0016] Embodiment 8. The system of any one of Embodiments 1 to 6, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a polypeptide.

[0017] Embodiment 9. The system of Embodiment 8, wherein the polypeptide is a peptide or a single chain antibody.

[0018] Embodiment 10. The system of any one of Embodiments 1 to 9, wherein the extracellular binding domain is a single chain Fv (scFv).

[0019] Embodiment 11. The system of any one of Embodiments 1 to 10, wherein the engineered receptor is a chimeric antigen receptor (CAR).

[0020] Embodiment 12. The system of any one of Embodiments 1 to 11, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4- IBB signaling domain and a CD28 signaling domain.

[0021] Embodiment 13. The system of Embodiment 12, wherein the costimulatory signaling domain is a 4-1BB signaling domain.

[0022] Embodiment 14. The system of any one of Embodiments 11 to 13, wherein the engineered cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

[0023] Embodiment 15. The system of any one of Embodiments 1 to 10, wherein the intracellular signaling domain is derived from an enzyme-linked cellular receptor.

[0024] Embodiment 16. The system of Embodiment 15, wherein the enzyme-linked cellular receptor is selected from the group consisting of an antigen receptor, a cytokine receptor, and a growth factor receptor. [0025] Embodiment 17. The system of Embodiment 16, wherein the enzyme-linked cellular receptor is the antigen receptor.

[0026] Embodiment 18. The system of Embodiment 16, wherein the antigen receptor is a T cell receptor.

[0027] Embodiment 19. The system of any one of Embodiments 1 to 10, wherein the engineered receptor is a synNotch receptor in which the intracellular signaling domain comprises a Notch transcriptional regulator that is released from the cell membrane upon engagement of the extracellular binding domain with the universal epitope.

[0028] Embodiment 20. The system of any one of Embodiments 1 to 19, wherein the encoded therapeutic protein is selected from the group consisting of an antibody, a second cell surface receptor, a soluble receptor, a cytokine, a chemokine, and a growth factor.

[0029] Embodiment 21. The system of Embodiment 20, wherein the antibody is a bispecific antibody.

[0030] Embodiment 22. The system of Embodiment 20 or 21, wherein the antibody is a single-chain antibody.

[0031] Embodiment 23. The system of Embodiment 21, wherein the bispecific antibody is a bispecific T cell engager.

[0032] Embodiment 24. The system of Embodiment 20, wherein the second cell surface receptor is a second engineered receptor.

[0033] Embodiment 25. The system of Embodiment 20, wherein the antibody is an immune checkpoint inhibitor.

[0034] Embodiment 26. The system of Embodiment 20, wherein the chemokine is selected from the group consisting of CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL16, CCL3, CCL4, and CCL5.

[0035] Embodiment 27. The system of Embodiment 20, wherein the cytokine is selected from the group consisting of interleukin-2 (IL-2), interleukin-4 (IL-4), interferonalpha (IFN-a), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), and interleukin-21 (IL-21).

[0036] Embodiment 28. The system of any one of Embodiments 1 to 10 and 15 to 19, wherein the therapeutic protein is an immunosuppressive therapeutic protein. [0037] Embodiment 29. The system of Embodiment 28, wherein the immunosuppressive therapeutic protein is selected from the group consisting of CTLA-4-Fc, TNFR-Fc, and an anti-TNFa antibody.

[0038] Embodiment 30. The system of any one of Embodiments 1 to 27, wherein the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment.

[0039] Embodiment 31. The system of Embodiment 30, wherein the universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tumor microenvironment.

[0040] Embodiment 32. The system of Embodiment 31, wherein the mask comprises a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide.

[0041] Embodiment 33. The system of any one of Embodiments 30 to 32, wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

[0042] Embodiment 34. The system of any one of Embodiments 30 to 32, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide.

[0043] Embodiment 35. The system of Embodiment 34, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of X{Ar}XCPY{G/D}LC{Ar}IX (SEQ ID NO: 1), wherein X = any amino acid, Ar = aromatic amino acid, and D/G = Gly or Asp; XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid; and NHGCYNSYGVPYCDYS (SEQ ID NO:3).

[0044] Embodiment 36. The system of Embodiment 34, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of Ac- Y{DGl}C{HPr}{Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, Y(3-C1) = 3-chlorotyrosine, and NH2 = C terminus amidation; and Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID NO: 5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation. [0045] Embodiment 37. The system of any one of Embodiments 30 to 32, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a collagen-binding peptide.

[0046] Embodiment 38. The system of Embodiment 37, wherein the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of (GP{HPr})?- (D)c, (SEQ ID NO:6), wherein HPr = hydroxyproline; (G{P(4-Fl)}{Hpr})_n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9; (GPP)s (SEQ ID NO: 8); (P{HPr}G)₇ (SEQ ID NO: 9), wherein HPr = hydroxyproline; TLTYTWS (SEQ ID NOTO); WYRGRL (SEQ ID NO: 11); TKKLTLRT (SEQ ID NO: 12); TKKTLRT (SEQ ID NO: 13); LRELTLNNN (SEQ ID NO: 14); and LRELHLNNN (SEQ ID NO: 15).

[0047] Embodiment 39. The system of any one of Embodiments 11 to 14, wherein the encoded therapeutic protein is a second CAR.

[0048] Embodiment 40. The system of Embodiment 39, wherein the second CAR specifically recognizes a tumor-associated antigen.

[0049] Embodiment 41. The system of any one of Embodiments 11 to 14, 39, and 40, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

[0050] Embodiment 42. The system of any one of Embodiments 1 to 41, wherein the polymer is a synthetic polymer.

[0051] Embodiment 43. The system of Embodiment 42, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units.

[0052] Embodiment 44. The system of Embodiment 43, wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

[0053] Embodiment 45. The system of Embodiment 43 or 44, wherein the polymer is a block copolymer.

[0054] Embodiment 46. The system of Embodiment 43 or 44, wherein the polymer is a statistical copolymer. [0055] Embodiment 47. A method for expressing a therapeutic protein at the site of a tissue or cell of interest in a subject, the method comprising: administering to a subject an effective regimen of

(i) a universal epitope, and

(b) an engineered cell comprising

(B) an intracellular signaling domain derived from a cell surface receptor, and

(ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the intracellular signaling domain upon engagement of the extracellular binding domain with the universal epitope; wherein the polymer specifically binds to the component of the tissue or cell of interest in the subject, thereby localizing the polymer to the site of the tissue or cell, and wherein the engineered receptor specifically binds to the universal epitope of the localized polymer, thereby inducing expression of the therapeutic protein at the site of the tissue or cell of interest.

[0056] Embodiment 48. The method of Embodiment 47, wherein the universal epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope. [0057] Embodiment 49. The method of Embodiment 47 or 48, wherein the universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tissue or cell of interest.

[0058] Embodiment 50. The method of Embodiment 49, wherein the mask comprises a self-immolative linker.

[0059] Embodiment 51. The method of Embodiment 50, wherein the self-immolative linker comprises a difluorophenyl ester or a p-aminobenzylalchohol.

[0060] Embodiment 52. The method of Embodiment 50, wherein the universal epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

[0061] Embodiment 53. The method of any one of Embodiments 47 to 52, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide.

[0062] Embodiment 54. The method of any one of Embodiments 47 to 52, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a polypeptide.

[0063] Embodiment 55. The method of Embodiment 54, wherein the polypeptide is a peptide or a single chain antibody.

[0064] Embodiment 56. The method of any one of Embodiments 47 to 55, wherein the extracellular binding domain is a single chain Fv (scFv).

[0065] Embodiment 57. The method of any one of Embodiments 47 to 56, wherein the engineered receptor is a chimeric antigen receptor (CAR).

[0066] Embodiment 58. The method of any one of Embodiments 47 to 57, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4-1BB signaling domain and a CD28 signaling domain.

[0067] Embodiment 59. The method of Embodiment 58, wherein the costimulatory signaling domain is a 4-1 BB signaling domain.

[0068] Embodiment 60. The method of any one of Embodiments 57 to 59, wherein the engineered cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell. [0069] Embodiment 61. The method of any one of Embodiments 47 to 56, wherein the intracellular signaling domain is derived from an enzyme-linked cellular receptor.

[0070] Embodiment 62. The method of Embodiment 61, wherein the enzyme-linked cellular receptor is selected from the group consisting of an antigen receptor, a cytokine receptor, and a growth factor receptor.

[0071] Embodiment 63. The method of Embodiment 62, wherein the enzyme-linked cellular receptor is the antigen receptor.

[0072] Embodiment 64. The method of Embodiment 62, wherein the antigen receptor is a T cell receptor.

[0073] Embodiment 65. The method of any one of Embodiments 47 to 56, wherein the engineered receptor is a synNotch receptor in which the intracellular signaling domain comprises a Notch transcriptional regulator that is released from the cell membrane upon engagement of the extracellular binding domain with the universal epitope.

[0074] Embodiment 66. The method of any one of Embodiments 47 to 65, wherein the encoded therapeutic protein is selected from the group consisting of an antibody, a second cell surface receptor, a soluble receptor, a cytokine, a chemokine, and a growth factor.

[0075] Embodiment 67. The method of Embodiment 66, wherein the antibody is a bispecific antibody.

[0076] Embodiment 68. The method of Embodiment 66 or 67, wherein the antibody is a single-chain antibody.

[0077] Embodiment 69. The method of Embodiment 67, wherein the bispecific antibody is a bispecific T cell engager.

[0078] Embodiment 70. The method of Embodiment 66, wherein the second cell surface receptor is a second engineered receptor.

[0079] Embodiment 71. The method of Embodiment 66, wherein the antibody is an immune checkpoint inhibitor.

[0080] Embodiment 72. The system of Embodiment 66, wherein the chemokine is selected from the group consisting of CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL16, CCL3, CCL4, and CCL5. [0081] Embodiment 73. The system of Embodiment 66, wherein the cytokine is selected from the group consisting of interleukin-2 (IL-2), interleukin-4 (IL -4), interferonalpha (IFN-a), interleukin- 12 (IL-12), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

[0082] Embodiment 74. The method of any one of Embodiments 47 to 56 and 61 to 65, wherein the therapeutic protein is an immunosuppressive therapeutic protein.

[0083] Embodiment 75. The method of Embodiment 74, wherein the immunosuppressive therapeutic protein is selected from the group consisting of CTLA-4-Fc, TNFR-Fc, and an anti-TNFa antibody.

[0084] Embodiment 76. The method of any one of Embodiments 47 to 73, wherein the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment.

[0085] Embodiment 77. The method of Embodiment 76, wherein the universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tumor microenvironment.

[0086] Embodiment 78. The method of Embodiment 77, wherein the mask comprises a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide.

[0087] Embodiment 79. The method of any one of Embodiments 76 to 78, wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

[0088] Embodiment 80. The method of any one of Embodiments 76 to 78, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide.

[0089] Embodiment 81. The method of Embodiment 80, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of X{Ar}XCPY{G/D}LC{Ar}IX (SEQ ID NO:1), wherein X = any amino acid, Ar = aromatic amino acid, and D/G = Gly or Asp; XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid; and NHGCYNSYGVPYCDYS (SEQ ID NO:3).

[0090] Embodiment 82. The method of Embodiment 80, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of Ac- Y{DGl}C{HPr} {Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID N0:4), wherein Ac=N-terminus acetylation, DGl=D-glutamic acid, HPr=hydroxyproline, Y(3-C1) = 3-chlorotyrosine, and NH2 = C terminus amidation; and Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID NO: 5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation.

[0091] Embodiment 83. The method of any one of Embodiments 76 to 78, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a collagen-binding peptide.

[0092] Embodiment 84. The method of Embodiment 83, wherein the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of (GP{HPr})7- (D)₆ (SEQ ID NO:6), wherein HPr = hydroxyproline; (G{P(4-Fl)}{Hpr})_n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9; (GPP)s (SEQ ID NO: 8); (P{HPr}G)7 (SEQ ID NO: 9), wherein HPr = hydroxyproline; TLTYTWS (SEQ ID NO: 10); WYRGRL (SEQ ID NO: 11); TKKLTLRT (SEQ ID NO: 12); TKKTLRT (SEQ ID NO: 13); LRELTLNNN (SEQ ID NO: 14); and LRELHLNNN (SEQ ID NO: 15).

[0093] Embodiment 85. The method of any one of Embodiments 57 to 60, wherein the encoded therapeutic protein is a second CAR.

[0094] Embodiment 86. The method of Embodiment 85, wherein the second CAR specifically recognizes a tumor-associated antigen.

[0095] Embodiment 87. The method of any one of Embodiments 57 to 60, 85, and 86, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

[0096] Embodiment 88. The method of any one of Embodiments 47 to 87, wherein the polymer is a synthetic polymer.

[0097] Embodiment 89. The method of Embodiment 88, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units.

[0098] Embodiment 90. The method of Embodiment 89, wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA). [0099] Embodiment 91. The method of Embodiment 89 or 90, wherein the polymer is a block copolymer.

[0100] Embodiment 92. The method of Embodiment 89 or 90, wherein the polymer is a statistical copolymer.

[0101] Embodiment 93. The method of any one of Embodiments 47 to 92, wherein the polymer is administered before the engineered cell.

[0102] Embodiment 94. The method of any one of Embodiments 47 to 92, wherein the engineered cell is administered before the polymer.

[0103] Embodiment 95. A system for polymer-mediated activation of a CAR immune cell, the system comprising:

(a) a polymer comprising a plurality of repeating units, wherein the polymer comprises

(i) a universal chimeric antigen receptor (CAR) epitope, and

(ii) a moiety that specifically binds to an extracellular matrix (ECM) component of a tumor microenvironment; and

(b) a CAR immune cell comprising

(i) a CAR that specifically recognizes the universal CAR epitope, wherein the CAR is constitutively expressed by the CAR immune cell, and

(ii) a transgene comprising a polynucleotide encoding a therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the CAR upon engagement of CAR with the universal CAR epitope.

[0104] Embodiment 96. The system of Embodiment 95, wherein the universal CAR epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope.

[0105] Embodiment 97. The system of Embodiment 95 or 96, wherein the universal CAR epitope is a masked epitope comprising a mask that is removed upon exposure to the tumor microenvironment. [0106] Embodiment 98. The system of Embodiment 97, wherein the mask comprises a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide.

[0107] Embodiment 99. The system of Embodiment 98, wherein the self-immolative linker comprises a difluorophenyl ester or a p-aminobenzylalchohol.

[0108] Embodiment 100. The system of Embodiment 97, wherein the universal CAR epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

[0109] Embodiment 101. The system of any one of Embodiments 95 to 100, wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

[0110] Embodiment 102. The system of any one of Embodiments 95 to 101, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide.

[OHl] Embodiment 103. The system of any one of Embodiments 95 to 101, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a polypeptide.

[0112] Embodiment 104. The system of any one of Embodiment 103, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a peptide or a single chain antibody.

[0113] Embodiment 105. The system of Embodiment 103, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a fibrin- binding peptide.

[0114] Embodiment 106. The system of Embodiment 105, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of X{Ar}XCPY{G/D}LC{Ar}IX (SEQ ID NO:1), wherein X = any amino acid, Ar = aromatic amino acid, and D/G = Gly or Asp; XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid; and NHGCYNSYGVPYCDYS (SEQ ID NO:3). [0115] Embodiment 107. The system of Embodiment 105, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of Ac- Y{DGl}C{HPr}{Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac=N-terminus acetylation, DGl=D-glutamic acid, HPr=hydroxyproline, Y(3-C1) = 3-chlorotyrosine, and NH2 = C terminus amidation; and Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID NO:5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation.

[0116] Embodiment 108. The system of Embodiment 103, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a collagen- binding peptide.

[0117] Embodiment 109. The system of Embodiment 108, wherein the collagen- binding peptide comprises an amino acid sequence selected from the group consisting of (GP{HPr})7-(D)6 (SEQ ID NO:6), wherein HPr = hydroxyproline; (G{P(4-Fl)}{Hpr})_n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9; (GPP)s (SEQ ID NO:8); (P{HPr}G)? (SEQ ID NO:9), wherein HPr = hydroxyproline; TLTYTWS (SEQ ID NO:10); WYRGRL (SEQ ID NO:11); TKKLTLRT (SEQ ID NO:12); TKKTLRT (SEQ ID NO: 13); LRELTLNNN (SEQ ID NO: 14); and LRELHLNNN (SEQ ID NO:15).

[0118] Embodiment 110. The system of any one of Embodiments 95 to 109, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4- IBB signaling domain and a CD28 signaling domain.

[0119] Embodiment 111. The system of Embodiment 110, wherein the costimulatory signaling domain is a 4-1BB signaling domain.

[0120] Embodiment 112. The system of any one of Embodiments 95 to 111, wherein the encoded therapeutic protein is a second CAR.

[0121] Embodiment 113. The system of Embodiment 112, wherein the second CAR specifically recognizes a tumor-associated antigen.

[0122] Embodiment 114. The system of Embodiment 113, wherein the second CAR is a bispecific CAR that specifically recognizes two different tumor-associated antigens. [0123] Embodiment 115. The system of Embodiment 112, wherein the second CAR specifically recognizes a second universal CAR epitope.

[0124] Embodiment 116. The system of any one of Embodiments 95 to 111, wherein the encoded therapeutic protein is an antibody.

[0125] Embodiment 117. The system of Embodiment 116, wherein the antibody is a bispecific antibody.

[0126] Embodiment 118. The system of Embodiment 116 or 117, wherein the antibody is a single-chain antibody.

[0127] Embodiment 119. The system of Embodiment 117, wherein the bispecific antibody is a bispecific T cell engager.

[0128] Embodiment 120. The system of Embodiment 1 16, wherein the antibody is an immune checkpoint inhibitor.

[0129] Embodiment 121. The system of Embodiment 120, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4, anti-PD-l/PD-Ll, and a combination thereof.

[0130] Embodiment 122. The system of any one of Embodiments 95 to 111, wherein the encoded therapeutic protein is an immunostimulatory cytokine.

[0131] Embodiment 123. The system of Embodiment 122, wherein the immunostimulatory cytokine is selected from the group consisting of interleukin-2 (IL-2), interferon-alpha (IFN-a), interleukin-12 (IL-12), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

[0132] Embodiment 124. The system of any one of Embodiments 95 to 123, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

[0133] Embodiment 125. The system of any one of Embodiments 95 to 124, wherein the CAR immune cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

[0134] Embodiment 126. The system of Embodiment 125, wherein the CAR immune cell is the CAR T cell.

[0135] Embodiment 127. The system of any one of Embodiments 95 to 126, wherein the polymer is a synthetic polymer. [0136] Embodiment 128. The system of Embodiment 127, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units.

[0137] Embodiment 129. The system of Embodiment 128, wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

[0138] Embodiment 130. The system of Embodiment 128 or 129, wherein the polymer is a block copolymer.

[0139] Embodiment 131. The system of Embodiment 128 or 129, wherein the polymer is a statistical copolymer.

[0140] Embodiment 132. A method for treating a solid tumor cancer, the method comprising: administering to a subject having the solid tumor cancer an effective regimen of

(i) a universal chimeric antigen receptor (CAR) epitope, and

(ii) a moiety that specifically binds to an extracellular matrix (ECM) component of the tumor microenvironment; and

(b) a CAR immune cell comprising

(ii) a transgene comprising a polynucleotide encoding an anticancer therapeutic protein, wherein the polynucleotide is operatively linked to an inducible promoter that is driven by an activation signal from the CAR upon engagement of CAR with the universal CAR epitope.

[0141] Embodiment 133. The method of Embodiment 132, wherein the universal CAR epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope. [0142] Embodiment 134. The method of Embodiment 132 or 133, wherein the universal CAR epitope is a masked epitope comprising a mask that is removed upon exposure to the tumor microenvironment.

[0143] Embodiment 135. The method of Embodiment 134, wherein the mask comprises a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide.

[0144] Embodiment 136. The method of Embodiment 135, wherein the self- immolative linker comprises a difluorophenyl ester or a p-aminobenzylalchohol.

[0145] Embodiment 137. The method of Embodiment 134, wherein the universal CAR epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

[0146] Embodiment 138. The method of any one of Embodiments 132 to 137, wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

[0147] Embodiment 139. The method of any one of Embodiments 132 to 138, wherein the polypeptide that specifically binds to the ECM component is a peptide or a single chain antibody.

[0148] Embodiment 140. The method of any one of Embodiments 132 to 137, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide.

[0149] Embodiment 141. The method of Embodiment 140, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of X{Ar]XCPY{G/D]LC{Ar}IX (SEQ ID NO:1), wherein X = any amino acid, Ar = aromatic amino acid, and D/G = Gly or Asp; XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid; and NHGCYNSYGVPYCDYS (SEQ ID NO:3).

[0150] Embodiment 142. The method of Embodiment 140, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of Ac- Y{DGl}C{HPr}{Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac=N-terminus acetylation, DGl=D-glutamic acid, HPr=hydroxyproline, Y(3-C1) = 3-chlorotyrosine, and NH2 = C terminus amidation; and Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID NO:5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation.

[0151] Embodiment 143. The method of any one of Embodiments 132 to 137, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a collagen-binding peptide.

[0152] Embodiment 144. The method of Embodiment 143, wherein the collagen- binding peptide comprises an amino acid sequence selected from the group consisting of (GP{HPr})7-(D)6 (SEQ ID NO:6), wherein HPr = hydroxyproline; (G{P(4-Fl)}{Hpr})_n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9; (GPP)s (SEQ ID NO:8); (P{HPr}G)₇ (SEQ ID NO:9), wherein HPr = hydroxyproline; TLTYTWS (SEQ ID NOTO); WYRGRL (SEQ ID NO: 11); TKKLTLRT (SEQ ID NO: 12); TKKTLRT (SEQ ID NO: 13); LRELTLNNN (SEQ ID NO: 14); and LRELHLNNN (SEQ ID NO: 15).

[0153] Embodiment 145. The method of any one of Embodiments 132 to 144, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4- IBB signaling domain and a CD28 signaling domain.

[0154] Embodiment 146. The method of Embodiment 145, wherein the costimulatory signaling domain is a 4-1BB signaling domain.

[0155] Embodiment 147. The method of any one of Embodiments 132 to 146, wherein the encoded anticancer therapeutic protein is selected from the group consisting of (i) a second CAR that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells, (ii) an antibody that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells, (iii) an immune checkpoint inhibitor, and (iv) an immunostimulatory cytokine.

[0156] Embodiment 148. The method of Embodiment 147, wherein the encoded therapeutic protein is the second CAR that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells. [0157] Embodiment 149. The method of Embodiment 148, wherein the second CAR is a bispecific CAR that specifically recognizes two different tumor-associated antigens.

[0158] Embodiment 150. The method of Embodiment 147, wherein the encoded therapeutic protein is the antibody the specifically recognizes a tumor-associated antigen expressed by the solid tumor cells.

[0159] Embodiment 151. The method of Embodiment 150, wherein the antibody is a bispecific antibody.

[0160] Embodiment 152. The method of Embodiment 150 or 151, wherein the antibody is a single-chain antibody.

[0161] Embodiment 153. The method of Embodiment 151, wherein the bispecific antibody is a bispecific T cell engager.

[0162] Embodiment 154. The method of Embodiment 147, wherein the encoded therapeutic protein is the immune checkpoint inhibitor.

[0163] Embodiment 155. The method of Embodiment 154, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4, anti-PD-l/PD-Ll, and a combination thereof.

[0164] Embodiment 156. The method of Embodiment 147, wherein the encoded anticancer therapeutic protein is an immunostimulatory cytokine selected from the group consisting of interleukin-2 (IL-2), interferon-alpha (IFN-a), interleukin- 12 (IL-12), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

[0165] Embodiment 157. The method of any one of Embodiments 132 to 156, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

[0166] Embodiment 158. The method of any one of Embodiments 132 to 157, wherein the polymer is administered before the CAR immune cell.

[0167] Embodiment 159. The method of any one of Embodiments 132 to 157, wherein the CAR immune cell is administered before the polymer.

[0168] Embodiment 160. The method of any one of Embodiments 132 to 159, wherein the solid tumor cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular carcinoma, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, basal cell carcinoma, cutaneous squamous cell carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, cervical cancer, endometrial carcinoma, esophageal carcinoma, gastric carcinoma, and Merkel cell carcinoma.

[0169] Embodiment 161. The method of any one of Embodiments 132 to 160, wherein the CAR immune cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

[0170] Embodiment 162. The method of Embodiment 161, wherein the CAR immune cell is the CAR T cell.

[0171] Embodiment 163. The method of any one of Embodiments 132 to 162, wherein the polymer is a synthetic polymer.

[0172] Embodiment 164. The method of Embodiment 163, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units.

[0173] Embodiment 165. The method of Embodiment 164, wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

[0174] Embodiment 166. The method of Embodiment 164 or 165, wherein the polymer is a block copolymer.

[0175] Embodiment 167. The method of Embodiment 164 or 165, wherein the polymer is a statistical copolymer.

[0176] Embodiment 168. The method of any one of Embodiments 47 to 94, wherein the effective regimen comprises administering a first dose of the polymer to activate engineered receptor signaling, wherein the activation of engineered receptor signaling is subsequently decreased or blocked, and administering at least a second dose of the polymer to reactivate the engineered receptor signaling following the decreased or blocked activation.

[0177] Embodiment 169. The method of any one of Embodiments 47 to 94 and 168, further comprising administering an agent that inhibits or blocks the engineered receptor activation signal. [0178] Embodiment 170. The method of any one of Embodiments 132 to 167, wherein the effective regimen comprises administering a first dose of the polymer to activate CAR signaling, wherein the activation of CAR signaling is subsequently decreased or blocked, and administering at least a second dose of the polymer to reactivate the CAR signaling following the decreased or blocked activation.

[0179] Embodiment 171. The method of any one of Embodiments 132 to 167 and 169, further comprising administering an agent that inhibits or blocks the CAR activation signal.

[0180] Embodiment 172. The system of any one of Embodiments 1 to 46, wherein the universal epitope is pendant to one or more of the repeating units forming the polymer backbone.

[0181] Embodiment 173. The system of any one of Embodiments 1 to 46, wherein the universal epitope is linked to the polymer at at least one of the polymer termini.

[0182] Embodiment 174. The system of any one of Embodiments 1 to 46, 172, and 173, wherein the moiety that specifically binds to the component of tissue or cell of interest is pendant to one or more of the repeating units forming the polymer backbone.

[0183] Embodiment 175. The system of any one of Embodiments 1 to 46, 172, and 173, wherein the moiety that specifically binds to the component of tissue or cell of interest is linked to the polymer at at least one of the polymer termini.

[0184] Embodiment 176. The method of any one of Embodiments 47 to 94, wherein the universal epitope is pendant to one or more of the repeating units forming the polymer backbone.

[0185] Embodiment 177. The method of any one of Embodiments 47 to 94, wherein the universal epitope is linked to the polymer at at least one of the polymer termini.

[0186] Embodiment 178. The method of any one of Embodiments 47 to 94, 176, and 177, wherein the moiety that specifically binds to the component of tissue or cell of interest is pendant to one or more of the repeating units forming the polymer backbone.

[0187] Embodiment 179. The method of any one of Embodiments 47 to 94, 176, and 177, wherein the moiety that specifically binds to the component of tissue or cell of interest is linked to the polymer at at least one of the polymer termini. [0188] Embodiment 180. The system of any one of Embodiments 95 to 131, wherein the universal CAR epitope is pendant to one or more of the repeating units forming the polymer backbone.

[0189] Embodiment 181. The system of any one of Embodiments 95 to 131, wherein the universal CAR epitope is linked to the polymer at at least one of the polymer termini.

[0190] Embodiment 182. The system of any one of Embodiments 95 to 131, 180, and 181, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is pendant to one or more of the repeating units forming the polymer backbone.

[0191] Embodiment 183. The system of any one of Embodiments 95 to 131, 180, and 181, wherein the moiety that specifically binds to the ECM component of tumor microenvironment is linked to the polymer at at least one of the polymer termini.

[0192] Embodiment 184. The method of any one of Embodiments 132 to 167, wherein the universal CAR epitope is pendant to one or more of the repeating units forming the polymer backbone.

[0193] Embodiment 185. The method of any one of Embodiments 132 to 167, wherein the universal CAR epitope is linked to the polymer at at least one of the polymer termini.

[0194] Embodiment 186. The method of any one of Embodiments 132 to 167, 184, and 185, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is pendant to one or more of the repeating units forming the polymer backbone.

[0195] Embodiment 187. The method of any one of Embodiments 132 to 167, 184, and 185, wherein the moiety that specifically binds to the ECM component of tumor microenvironment is linked to the polymer at at least one of the polymer termini.

[0196] Embodiment 188. The system of Embodiment 103, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a peptide that binds to an N-terminal fibronectin type I domain.

[0197] Embodiment 189. The system of Embodiment 188, wherein the peptide that binds to an N-terminal fibronectin type I domain comprises the amino acid sequence GGGQVTTESNLVEFDEESTKGIVTGAVSDHTTVEDTK (SEQ ID NO: 18). [0198] Embodiment 190. The method of any one of Embodiments 132 to 137, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a peptide that binds to an N-terminal fibronectin type I domain.

[0199] Embodiment 191. The method of Embodiment 190, wherein the peptide that binds to an N-terminal fibronectin type I domain comprises the amino acid sequence GGGQVTTESNLVEFDEESTKGIVTGAVSDHTTVEDTK (SEQ ID NO: 18).

[0200] These and other aspects and embodiments will become evident upon reference to the following detailed description and the attached drawings.

DEFINITIONS

[0201] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0202] The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise.

[0203] A “tissue or cell of interest,” as used herein, means any tissue or cell that may be targeted for localization of an engineered cell and delivery of an encoded therapeutic protein using a system as disclosed herein. A “tissue or cell of interest” may also be referred to herein as a “target tissue or cell.” A moiety that specifically binds to a component of a target tissue or cell may also be referred to herein as a “targeting moiety.”

[0204] As used herein, the term “universal epitope” means a target molecule specifically recognized by the engineered receptor and that is orthogonal to a mammalian biological system (i.e., does not interfere with the native biological processes of mammalian cells or tissues). The term “specifically recognizes,” in reference to an engineered receptor and universal epitope, means that the extracellular binding domain of the receptor specifically binds to the universal epitope. The epitope is termed “universal” as one epitope can be used to target engineered cells to different target tissues and cells using different targeting moi eties.

[0205] The term “specifically binds,” as used herein, refers to the ability of a targeting moiety or a receptor extracellular binding domain to bind to its corresponding target (respectively, a component of a target tissue or cell, or a displayed universal epitope) with a dissociation constant (Kd) of at most about 1 * 1 O'⁶ M, and/or to bind to its target with an affinity that is at least about two-fold greater than its affinity for a nonspecific target molecule. In some embodiments, a targeting moiety or a receptor extracellular binding domain specifically binds to its corresponding target with a Kd of at most about l * 10’⁷ M, at most about l * 10’⁸ M, at most about 1 x 10’⁹ M, or at most about 1 x IO’¹⁰ M.

[0206] A “polymer,” as used herein in reference to a polymer displaying a universal epitope and a targeting moiety, refers to any macromolecule having a backbone produced, whether naturally or synthetically, by polymerization of small molecule monomers. Polymer subunits derived from the polymerized monomers are also referred to herein as “repeating units.”

[0207] A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 50 amino acid residues may also be referred to as “peptides.”

[0208] As used herein, a “protein” is a macromolecule comprising one or more polypeptide chains. A protein (e.g., therapeutic protein) may also comprise non-peptidic components, such as carbohydrate groups, which may be added to a protein by the cell in which the protein is produced.

[0209] The term “engineered receptor,” as used herein, is a fusion protein having a structure containing a target-specific extracellular domain (“extracellular binding domain”), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain is capable of generating a signal to activate an inducible promoter driving expression of a therapeutic transgene within a cell expressing the engineered receptor. In addition, the intracellular signaling domain may work for other effector functions of the cell expressing the engineered receptor. In some embodiments, an engineered receptor is a “chimeric antigen receptor” (CAR) in which the intracellular signaling domain comprises a domain for transmitting a signal mediated by TCR complex (also referred to as “first domain” for convenience), and may further comprise a domain for transmitting a co- stimulatory signal (also referred to as “second domain” for convenience). A cell expressing an engineered receptor is referred to herein as an “engineered cell,” and immune cells expressing a CAR are referred to herein as a “CAR immune cell.” [0210] The term “antibody,” as used herein, refers to an immunoglobulin molecule, or a fragment and/or engineered variant thereof, which has the ability to specifically bind to an antigen. The term “antibody” includes intact monoclonal antibodies and antigen-binding antibody fragments such as, e.g., F(ab’)2 and Fab fragments. Genetically engineered intact antibodies and fragments, such as chimeric antibodies, humanized antibodies, single-chain Fv fragments, single-chain antibodies, diabodies, minibodies, and the like are also included. Thus, the term “antibody” is used expansively to include any protein that comprises an antigenbinding site and is capable of binding to its antigen.

[0211] An “antigen-binding site” is that portion of an antibody that is sufficient to bind to its antigen. The minimum such region is typically an immunoglobulin variable domain or fragment thereof, or a genetically engineered variant of an immunoglobulin variable domain or fragment thereof. Single-domain binding sites can be generated from camelid antibodies (see Muyldermans and Lauwerey s, J. Mol. Recog. 12:131-140, 1999; Nguyen et al. , EMBO J. 19:921-930, 2000) or from immunoglobulin heavy chain variable (VH) regions of other species to produce single-domain antibodies (“dAbs”; see Ward et al., Nature 341:544-546, 1989; US Patent No. 6,248,516 to Winter et al.). In certain variations, an antigen-binding site is a polypeptide region having only two complementarity determining regions (CDRs) of a naturally or non-naturally (e.g., mutagenized) occurring heavy chain variable domain or light chain variable domain, or combination thereof (see, e.g., Pessi et al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol. 25:921-929, 2007).

[0212] As used herein, the term “single-chain antibody” refers to an antibody having an antigen-binding site contained within a single polypeptide chain (e.g., the variable regions from both immunoglobulin heavy and light chains — also referred to herein as the immunoglobulin “VH” and “VL” regions — within a single polypeptide chain). The term “single-chain Fv” refers to a single-chain antibody that comprises the VH and VL regions but lacks immunoglobulin constant regions. In general, a single-chain Fv further comprises a polypeptide linker between the VH and VL regions, which enables it to form the desired structure that allows for antigen binding. Single-chain antibodies are discussed in detail by, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113 (Rosenburg and Moore eds., Springer-Verlag, New York, 1994), pp. 269-315. (See also International PCT Publication No. WO 88/01649; U.S. Patent Nos. 4,946,778 and 5,260,203; Bird etal., Science 242:423-426, 1988.) Single-chain antibodies can also be bi-specific and/or humanized.

[0213] The term “alternative scaffold protein” refers to a non-antibody protein in which one or more regions may be diversified to produce one or more binding domains that specifically bind to a target molecule. In some embodiments, the binding domain binds the target molecule with specificity and affinity similar to that of an antibody. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), the 0-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), gamma-B- crystallin/ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and Avimers. Additional information on alternative scaffolds is provided in Binz et al., Nat. Biotechnol. 23: 1257-1268, 2005; Skerra, Current Opin. in Biotech. 18:295-304, 2007; and Silacci etal., J. Biol. Chem. 289: 14392-14398, 2014.

[0214] The term “tumor-associated antigen” refers to proteins, glycoproteins, or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0215] The term “promoter” is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription.

[0216] “Operatively linked” means that two or more entities are joined together such that they function in concert for their intended purposes. When referring to DNA segments, the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator.

[0217] The term “patient” or “subject,” in the context of delivering a therapeutic protein at the site of a tissue or cell of interest or treating a disease or disorder (e.g., a solid tumor cancer) as described herein, includes mammals such as, for example, humans and other primates. The term also includes domesticated animals such as, e.g., cows, hogs, sheep, horses, dogs, and cats.

[0218] The terms “treat” and “treatment” are used broadly to denote therapeutic and prophylactic interventions that favorably alter a pathological state. [0219] The term “effective regimen,” as used herein, means a combination of amounts of a polymer and an engineered cell being administered and dosage frequency adequate to accomplish expression of a therapeutic protein at the site of a tissue or cell of interest or treatment of a disease or disorder (e.g., a solid tumor cancer) in accordance with the present disclosure.

[0220] When a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%.

[0221] Where aspects or embodiments of the present disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. The present disclosure also envisages the explicit exclusion of one or more of any of the group members as embodiments.

DESCRIPTION OF THE DRAWINGS

[0222] FIG. l is a schematic depicting an embodiment of polymer-mediated activation of engineered cells in accordance with the present disclosure. This embodiment, also referred to herein situ Mobilization: Polymer-Activated Cell Therapies” (IMPACT), relies on PolySTAT to induce regulated transgene expression. IMPACT therapy payload is only delivered after the engineered cells engage their ligand on the PolySTAT polymer. (A) Cells remain in a quiescent state regardless of their local environment. (B) PolySTAT, once administered, binds to fibrin in tumor microenvironments and presents the cognate antigen to the engineered cells. (C) This interaction stimulates the cells and drives expression of an iSynPro-restricted transgene, such as an mCherry:ffluc reporter protein in this proof-of- concept demonstration.

[0223] FIGs. 2A-2G show stringent activation-dependent transcription from the iSynPro promoter in cCARiCherry T cells. cCARiCherry T cells demonstrate restricted payload expression until their CARs or TCRs are engaged. All results are representative of two donors. FIG. 2A: Design of the cCARiCherry Piggybac construct. The construct includes an iSynPro-driven mCherry:ffluc fusion protein and an EFla promoter-driven anti-FITC(E2- mut2) CAR. FIG. 2B: Diagram showing restricted payload expression until CAR engages ligand. FIG. 2C: Purity of CAR+ T cells (CD19t expression marker) was determined by flow cytometry. FIG. 2D: Flow cytometry shows that K562 P cells emit strong FL signal when labeled with FL-PLE. FIGs. 2E-2G: cCARiCherry or mock T cells were stimulated with K562 P, FL-PLE-labeled K562 P, or K562 OKT3 cells for 24 hours. FIG. 2E: CD69 expression was upregulated in mock and cCARiCherry T cells incubated with positive control K562 OKT3 cells. CD69 was comparably upregulated in cCARiCherry T cells, but not mock T cells, incubated with FL-PLE-labeled K562 P cells. FIG. 2F: Expression of mCherry was upregulated in cCARiCherry T cells when stimulated with FL-PLE-labeled K562 cells and dependent on CAR expression. CAR-negative cells did not produce mCherry when stimulated. FIG. 2G: Expression of mCherry was upregulated comparably in cCARiCherry T cells stimulated with K562 OKT3 cells and FL-PLE-labeled K562 cells.

[0224] FIGs. 3A-3D show impact of fluorescein orientation and linker design on anti- FL antibody binding to PolySTAT. PolySTAT was modified to improve anti-fluorescein antibody binding. FIG. 3 A: Polymer structures for Poly STATs containing AEMA-FL monomer and fluorescein O-methacrylate (FMA). PS-ELISA outputs demonstrate that AEMA-FL PolySTAT provided a better platform for anti-FL antibody binding than FMA- PolySTAT in a PolySTAT -ELISA. FIG. 3B: Polymer structures for AEMA-FL PolySTATs containing 2-hydroxylethyl methacrylate (HEMA) or glycerol monomethacrylate (GmMA) backbone monomers. Polymers with GmMA have enhanced anti-FL antibody binding compared with polymers with HEMA monomers. FIG. 3C: PolySTATs and non-fibrin- binding PolySCRMs with 2% (original percentage), 4% and 10% AEMA-FL monomers in statistical polymer structure. PolySTAT -ELISA indicated that increasing FL content did not increase antibody binding signal. FIG. 3D: Evaluation of PolySTATs and non-fibrin-binding PolySCRMs with 2%, 4%, and 10% AEMA-FL monomers in block copolymer structure. PolySTAT-ELISA suggest that increasing FL content had a negative effect on anti-FL antibody binding signal. For all bar graphs, n=3. Data presented are mean values +/- SD. *=p<0.01, **=p<0.001, ***=p<0.0001 .

[0225] FIGs. 4A-4C show expression of mCherry in cCARiCherry cells when stimulated with various AEMA-FL PolySTAT variants. cCARiCherry T cells are rapidly activated and drive payload expression in vitro when incubated on PolySTAT -fibrin gel. FIG. 4A: Red image means of gels measured by an IncuCyte every 2 hours for 20 hours demonstrate mCherry:ffluc production from cCARiCherry T cells incubated on different AEMA-FL- PolySTAT/fibrinogen gels. FIGs. 2B, 2C: After a 20-hour incubation on AEMA-FL- PolySTAT gels, cCARiCherry T cells were harvested and mCherry (FIG. 2B) and CD69 (FIG. 2C) expression were analyzed by flow cytometry.

[0226] FIGs. 5 A and 5B show in vivo activation of FL-specific CAR T cells with FL- PolySTAT for iSynPro reporter expression. FIG. 5A is a schematic diagram of cCARiCherry T cell activation experiment. Mock or cCARiCherry T cells were injected IV into NSG mice. Two days later, PBS, or lOmg/kg of 2% AEMA-FL-PolySTAT or PolySCRM were injected IV. Mice were then imaged every 24 hours for 7 days to record induced ffluc signal. FIG. 5B shows quantification of bioluminescence flux (photons/second). Results show significantly higher flux from cCARiCherry T cells in mice injected with AEMA-FL-PolySTAT than either negative control. A one-way ANOVA test was performed at each time point. Tukey’s HSD post-hoc analysis was then performed between each group. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. n=4 biologically independent replicates +/- SD.

[0227] FIGs. 6A-6D show activation of cCARiCherry T cells with FMA-PolySTAT. FIG. 6A: FMA-PolySTAT and fibrin binding protein chemical structures. FIG. 6B: Diagram depicting the gel fabrication process. Thrombin and salt solution was mixed with PolySTAT. Fibrinogen was then added, and the gels were cured for one hour. FIG. 6C: Diagram depicting the cell activation process. Cells were added onto solidified gels and incubated until harvested and analyzed. FIG. 6D: CD69 expression and mCherry production from mock or cCARiCherry T cells incubated on 5 pM, 10 pM, or 25 pM FMA-PolySTAT and FMA- PolySCRM gels for 20 hours. Results are for one donor.

[0228] FIG. 7 depicts synthesis of AEMA-FL monomer. 2-aminoethyl methacrylate hydrochloride was reacted with fluorescein isothiocyanate overnight at room temperature in DMSO with DIPEA. The monomer was then precipitated in 6% HC1 solution, filtered, and lyophilized.

[0229] FIGs. 8A-8C depict a synthesis scheme for statistical AEMA-FL PolySTAT.

[0230] FIGs 9A-9D depict a synthesis scheme for block copolymer AEMA-FL PolySTAT. [0231] FIG. 10 shows quantification of daily bioluminescence flux (photons/second) from mice injected IV with either polySTAT or PolySCRAM followed by cCARiCherry T cells 24 hours later. Results show significantly higher flux from cCARiCherry T cells in mice injected with AEMA-FL-PolySTAT than with AEMA-FL-Poly SCRAM control polymer which does not bind fibrin at numerous time points. A one-way unpaired T test with Welch’s correction was performed at each time point. *=p<0.05, n=3 biologically independent replicates +/- SD.

DETAILED DESCRIPTION

[0232] The present disclosure provides systems and methods to spatially restrict engineered cell activation to target cells and tissues, including spatially restricted delivery of a therapeutic payload to the target site. Specifically, a polymer displaying both (i) a universal epitope recognized by an engineered receptor and (ii) a targeting moiety that specifically binds to a target tissue or cell is used in combination with engineered cells that both constitutively express the engineered receptor and contain a transgene encoding a therapeutic protein, wherein the transgene is under the control of an inducible promoter driven by an activation signal from the engineered receptor. The disclosed systems and methods provide, e.g., for enhanced control over activation of engineered cells, including precise delivery of therapeutic agents to a target site while also minimizing off-target effects (e. ., off-target toxicities). The systems and methods of the present disclosure are particularly useful, for example, for enhancing the safety and specificity of CAR immune cell therapies such as, e.g., for the treatment of solid tumor cancers by targeting extracellular matrix (ECM) components of the tumor microenvironment. In addition, due to the modular nature of the disclosed system, different pieces of the system (e.g., targeting moiety, universal epitope, engineered receptor domains, and inducible therapeutic protein transgene) can be refined to address situational requirements of different disease conditions and individual patients. For example, the targeting moiety of the polymer may be used to target any of various different tissue or cell types involved in the pathology of a disease or disorder of interest, and a corresponding engineered cell designed to engage the universal epitope of the polymer may then be used to specifically express a relevant therapeutic protein at the target site to treat the disease or disorder. [0233] Accordingly, in one aspect, the present disclosure provides a system for polymer-mediated activation of an engineered cell. The system generally includes

(i) a universal epitope, and

(b) an engineered cell comprising

(B) an intracellular signaling domain derived from a cell surface receptor, and

[0234] Each of the universal epitope and the targeting moiety can be present at one or more positions on the polymer. For example, either of the universal epitope and the targeting moiety can be pendant to one or more of the repeating units forming the polymer backbone. Alternatively, either of the universal epitope and the targeting moiety can be linked to the polymer at at least one of the polymer termini. Any combination of the aforementioned linkages may be used. For example, in certain variations, the universal epitope and the targeting moiety are each independently pendant to one or more of the repeating units. In other variations, the universal epitope is linked at one of the polymer termini and the targeting moiety is linked at the other of the polymer termini. In other embodiments, the universal epitope is pendant to one or more of the repeating units and the targeting moiety is linked at one or both of the polymer termini. In yet other embodiments, the targeting moiety is pendant to one or more of the repeating units and the universal epitope is linked at one or both of the polymer termini. The preparation and properties of representative polymers having a pendant universal epitope and a pendant targeting moiety are described in Example 1 and FIGs. 3A-3D, 4A-4C, 6 A, 7, 8A-8C, and 9A-9D.

[0235] Universal epitopes suitable for use with engineered cells in accordance with the present disclosure include, e.g., epitopes used with known universal CAR immune cells. See, e.g., Kim et al., J. Am. Chem. Soc. 137:2832-2835, 2015; Urbanska et al., Cancer Res. 72: 1844-1852, 2012; Rodgers et al., Proc. Natl. Acad. Set. USA 113:E459-468, 2016; Minutolo et al., J. Am. Chem. Soc. 142:6554-6568, 2020. Particularly suitable universal epitopes include fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, AlexaFluor dyes, rhodamine derivatives, and peptide neo-epitopes.

[0236] In some variations of a system for polymer-mediated activation of an engineered cell as above, a universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tissue or cell of interest. In some embodiments, a mask includes a self-immolative linker such as, e.g., a difluorophenyl ester or a p-aminobenzylalchohol. In some embodiments wherein the component of the tissue or cell of interest is a component of a tumor microenvironment, a mask includes a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide. In certain specific variations wherein the universal epitope is fluorescein, a masked epitope includes an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

[0237] In some embodiments of a system as above, a moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide. In certain variations, a polypeptide moiety is a peptide or a single-chain antibody. In other variations, a polypeptide moiety is an alternative scaffold protein.

[0238] In certain embodiments of a system as above, the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment. In some such embodiments, the ECM component of the tumor microenvironment is selected from fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

[0239] In some embodiments of a system as above wherein the tissue or cell component is an ECM component, the moiety is a fibrin-binding peptide. Particularly suitable fibrin-binding peptides comprise an amino acid sequence selected from (i) X{Ar}XCPY{G/D]LC{Ar}IX (SEQ ID NO:1), wherein X = any amino acid, Ar = aromatic amino acid, and D/G = Gly or Asp; (ii) XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid; and (iii) NHGCYNSYGVPYCDYS (SEQ ID NO:3). See, e.g., Kolodziej et al., Bioconjugate Chem. 23:548-556, 2012. In some specific variations, a fibrin-binding peptide comprises an amino acid sequence selected from (i) Ac-Y{DGl}C{HPr}{Y(3- C1)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac = N-terminus acetylation, DG1 = D- glutamic acid, HPr = hydroxyproline, Y(3-C1) = 3 -chlorotyrosine, and NH2 = C terminus amidation; and (ii) Ac-W{DGl }C{HPr}WGLCWIQGK-NH2 (SEQ ID NO:5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation. Fibrin-binding peptides as above are cyclized via an intramolecular disulfide bond formed between two cysteine residues.

[0240] In other embodiments of a system as above wherein the tissue or cell component is an ECM component, the moiety is a collagen-binding peptide. In some such embodiments, the collagen-binding peptide binds to damaged collagen, type I collagen, denatured type IV collagen, or denatured type II collagen. Particularly suitable collagen-binding peptides comprise an amino acid sequence selected from (i) (GP{HPr})?-(D)6 (SEQ ID NO:6), wherein HPr = hydroxyproline (targeting damaged collagen); (ii) (G{P(4-Fl)}{Hpr})_n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9 (targeting damaged collagen); (iii) (GPP)s (SEQ ID NO:8) (targeting damaged collagen); (iv) (P{HPr]G)7 (SEQ ID NO:9), wherein HPr = hydroxyproline (targeting type I collagen); (v) TLTYTWS (SEQ ID NO: 10) (targeting denatured type IV collagen); (vi) WYRGRL (SEQ ID NO: 11) (targeting denatured type II collagen); (vii) TKKLTLRT (SEQ ID NO: 12) (targeting type I collagen); (viii) TKKTLRT (SEQ ID NO: 13) (targeting type I collagen); (ix) LRELTLNNN (SEQ ID NO: 14) (targeting type I collagen); and (x) LRELHLNNN (SEQ ID NO: 15) (targeting type I collagen). See, e.g., Cai et al., ACS Appl. Bio Mater. 3:7492-7499, 2020; Chattopadhyay et al., Org. Biomol. Chem. 10:5892-5897, 2012; Wei et al., J. Mater. Chem. B 8(44), 2020; Mo et al., Angew. Chem. Int. Ed. Engl. 45:2267-22670, 2006; Mueller et al., Mol. Cancer Res. 7:1078-1085, 2009; Rothenfluh et al., Nat. Mater. 7:248-254, 2008; Boone et al., Appl. Set. 13:3342, 2023.

[0241] In other embodiments of a system as above wherein the tissue or cell component is an ECM component, the moiety is a fibrin-fibronectin complex-binding peptide. A particularly suitable fibrin-fibronectin complex-binding peptide is a peptide comprising the amino acid sequence CREKA (SEQ ID NO: 16). See, e.g., Zhou et al., Nat. Commun. 6:7984, 2015.

[0242] In other embodiments of a system as above wherein the tissue or cell component is an ECM component, the moiety is a peptide that binds to the EBD domain of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, or heparan sulfate and heparin. In some such embodiments, moiety is a peptide comprising an amino acid sequence selected from (i) HSCSSPIQGSWTWENGKWTWKGIIRLEQQP (SEQ ID NO: 17) (targeting the EBD domain of fibronectin); (ii) GGGQVTTESNLVEFDEESTKGIVTGAVSDHTTVEDTK (SEQ ID NO: 18) (targeting the N-terminal fibronectin type I domains); (iii) FHKHKSPALSPVGGG (SEQ ID NO: 19) (targeting tenascin-C); and (iv) CARSKNKDC (SEQ ID NO:20) (targeting heparan sulfate and heparin). See, e.g., Jeon et al., Mol. Pharmaceutics 14:3772-3779, 2017; Arnoldini et al., Nat. Commun. 8: 1793, 2017; Kim et al., Mol. Cells 33:71 -77, 2012; Jarvinen and Ruoslahti, Am. J. Pathol., 171 :702-711, 2007.

[0243] The extracellular binding domain of the engineered receptor contains a recognition site that exhibits specific binding to the universal epitope. In certain variations, the extracellular binding domain comprises an antigen-binding site of an antibody. Particularly suitable extracellular binding domains contain an antigen-binding site of an antibody within a single polypeptide chain. In some specific embodiments, the extracellular binding domain is a single chain Fv (scFv) or a nanobody. In other variations, the extracellular binding domain is an alternative scaffold protein. In yet other variations, e.g., when the target is a cell surface receptor, the extracellular binding domain is a ligand or a counter-receptor extracellular domain (ECD) that binds to the receptor, or a binding domain derived from the ligand or counter-receptor ECD. [0244] For example, a single chain antibody (e.g., an scFv) having specificity for the universal epitope may be derived from a monoclonal antibody against the universal epitope. A monoclonal antibody for derivation of a single chain antibody can be, for example, a rodent (such as mouse, rat, or rabbit) antibody, a human antibody, or a humanized antibody. An scFv has a structure in which a light chain variable region (VL) and a heavy chain variable region (VH) of an immunoglobulin are connected via a peptide linker, and it retains the ability to bind to an antigen. Examples of peptide linkers include linkers composed of glycine and/or serine (e.g., GGS or GS linker). Glycine and serine are small in size, preventing the linker from forming a higher-order structure. The length of the linker is not particularly limited to any specific length. For example, a linker having five to 25 amino acid residues can be used. The length of the linker is typically eight to 25 amino acids and more typically 15 to 20 amino acids.

[0245] In some specific variations, the extracellular binding domain is an antifluorescein single chain antibody. Anti-fluorescein antibodies — including, for example, antifluorescein scFvs (e.g., FITC-E2) — and their use in the context of engineered receptors are generally known in the art and can be readily incorporated and adapted for use in accordance with the present disclosure (see, e.g., Ma et al., Proc. Natl. Acad. Set USA 113:E450-E458, 2016; Honegger et al., Protein Science 14:2537-2549, 2005; International Patent Application Publication No. WO 2019/144091 and WO 2019/156795).

[0246] Other suitable extracellular binding domains include, for example, antibodies (e.g., scFvs) that bind to peptide epitopes. Exemplary antibodies binding to peptide epitopes include mAb 5B9 (recognizing a 10 amino acid epitope) and mAb 7B6 (recognizing an 18 amino acid epitope. See Feldmann et al., Oncolmmunology 9: 1-15, 2020. These binding domains have been utilized in universal CARs, see id., and are readily adaptable for use in engineered receptors in accordance with the present disclosure, together with polymers presenting the 5B9 or 7B6 peptide epitope.

[0247] Still other suitable extracellular binding domains include, for example, antibodies (e.g., scFvs) that bind to haptens. Exemplary hapten-binding antibodies include antibodies specific for dinitrophenyl (DNP) (see Rong et al., J. Mol. Biol. 434: 167513, 2022; describing universal CARs against DNP) and 1 ,3 diketone (see Shabat et al., Proc. Natl. Acad. Sci. USA 98:7528-7533, 2001; describing use of the 38C2 antibody against 1,3 diketone). Engineered receptors presenting these antibodies can be readily used in accordance with the present disclosure, together with polymers displaying the corresponding hapten.

[0248] An engineered receptor further contains a transmembrane domain situated between the extracellular binding domain and the intracellular signaling domain. Suitable transmembrane domains include, for example, transmembrane domains of CD8, T cell receptor a or chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, GITR, and 4-1BB, to name a few. A transmembrane domain can also be an artificially constructed polypeptide. Any of the foregoing exemplary transmembrane domains are suitable for use in, e. ., a chimeric antigen receptor (CAR). In some preferred embodiments of a CAR, the transmembrane domain is the transmembrane domain of CD28.

[0249] In some embodiments of a system as above, the intracellular signaling domain is derived from an enzyme-linked cellular receptor such as, for example, an antigen receptor, a cytokine receptor, or a growth factor receptor. In some such embodiments wherein the intracellular signaling domain is derived from an antigen receptor, the antigen receptor is a T cell receptor.

[0250] In particular embodiments of a system as above wherein the intracellular signaling domain is derived from an antigen receptor, the engineered receptor is a chimeric antigen receptor (CAR). In some variations comprising a CAR, the engineered cell is a CAR immune cell (c. , a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell). In addition to transmitting an activation signal to the inducible promoter driving expression of the encoded therapeutic protein, the intracellular signaling domain of a CAR immune cell, upon engagement of the extracellular binding domain with its target, transmits a signal that allows the immune cell to exert its effector function. The CAR intracellular signaling domain typically comprises a domain for transmitting a signal mediated by TCR complex (also referred to as a “first signaling domain”), and may further comprise a domain for transmitting a costimulatory signal (also referred to as a “second signaling domain”). Examples of these domains include signaling domains of CD2, CD4, CD5, CD28, CD134, 4-1BB (CD137), GITR, CD27, 0X40, HVEM, CD3< FcsRIy, OX-40, and ICOS, to name a few. The first signaling domain is typically the signaling domain of CD3(^ or FceRIy, more typically the domain of CD3^. The second signaling domain is typically the signaling domain of CD28, 4- 1BB (CD137), CD2, CD4, CD5, CD134, OX-40, or ICOS, and more typically the signaling domain of CD28 or 4-1BB. The first signaling domain and the second domain are typically different domains connected in tandem. In some embodiments, the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4-1BB signaling domain and a CD28 signaling domain.

[0251] When a CAR intracellular signaling domain includes a first signaling domain and a second signaling domain, the first signaling domain and the second signaling domain may be connected in any way, but the second signaling domain is typically placed on the transmembrane domain side, since it is known that co-stimulation can be strongly transmitted in some cases when the first signaling domain (e.g., CD3Q is connected distally. The first signaling domain and the second signaling domain may be directly connected or may be connected by a peptide linker. The linker that connects the first signaling domain and the second signaling domain is not particularly limited to any specific length. For example, a linker having from two to 15 amino acid residues may be used.

[0252] Chimeric antigen receptors (CARs) and CAR immune cells are generally known in the art and are also described, for example, in Mazinani and Rahbarizadeh, Biomarker Research 10:70, 2022; June etal., Science 359: 1361, 2018; Majzner and Mackall, Nature Medicine 25: 1341, 2019; U.S. Patent Nos. 7,446,190 and 9,629,877; and International Patent Application Publication Nos. WO 2012/079000, WO 2017/019848, WO 2019/144091, WO 2020/205359, and WO 2022/125850.

[0253] In other embodiments of a system as above, the engineered receptor is a synthetic Notch (synNotch) receptor in which the intracellular signaling domain comprises a Notch transcriptional regulator that is released from the cell membrane upon engagement of the extracellular binding domain with the universal epitope. The use of chimeric forms of Notch as a platform for designing engineered receptors and cells is generally known in the art (see, e.g., Morsut et al. , Cell 164:780-791, 2016; Roybal et l., Cell 167:419-432.el6, 2016; Luo et al., Front. Oncol. 9: 1448, 2019), and synNotch receptors that specifically recognize a universal epitope may be readily used in accordance with systems and methods of the present disclosure.

[0254] The extracellular binding domain and the transmembrane domain of an engineered receptor may be connected via a spacer domain. A spacer domain may be used to promote the binding of the engineered receptor to its target. For example, as the spacer domain of, e.g., of a chimeric antigen receptor, an Fc region of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof may be used. For example, an Fc region of human IgG (e.g., human IgGl or human IgG4) may be used as a spacer domain. In some alternative variations, a part of the extracellular domain of CD28 and a part of the extracellular domain of CD8a may be used as a spacer domain. A spacer domain can also be provided between the transmembrane domain and the intracellular signal domain.

[0255] Suitable encoded therapeutic proteins for use with a system as above include, for example antibodies, cell surface receptors, soluble receptors, cytokines, chemokines, and growth factors. In some embodiments wherein the therapeutic protein in an antibody, the antibody is a bispecific antibody and/or a single-chain antibody. In some embodiments comprising a bispecific antibody, the antibody is a bispecific T cell engager. In other embodiments wherein the therapeutic protein is an antibody, the antibody is an immune checkpoint inhibitor such as, e.g., anti-CTLA-4, anti-PD-l/PD-Ll, or a combination thereof.

[0256] In some embodiments, the therapeutic protein is a cell surface receptor (also referred to herein as a “second” cell surface receptor merely for convenience to distinguish a cell surface receptor serving as the encoded therapeutic protein from the native cell surface receptor from which the engineered receptor intracellular signaling domain is derived). In some such variations, the therapeutic protein is a second engineered receptor, which may specifically recognize, e.g., a second universal epitope (which may be present, e.g., on a second polymer or intermediate adaptor molecule) or a target antigen expressed in a target tissue or cell. For example, in certain embodiments wherein the engineered receptor is a chimeric antigen receptor (CAR), the encoded therapeutic protein is a second CAR (e.g., a second CAR that specifically recognizes a tumor-associated antigen). [0257] In other embodiments, the therapeutic protein is a cytokine. In some such embodiments, the cytokine is an interleukin or an interferon. In certain variations, a cytokine is an immunostimulatory cytokine such as, e.g, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin- 12 (IL- 12), interferon-alpha (IFN-a), interleukin- 15 (IL- 15), or interleukin-21 (IL- 21).

[0258] In other embodiments, the therapeutic protein is a chemokine. In some such embodiments, the chemokine is CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL16, CCL3, CCL4, or CCL5

[0259] In some embodiments of a system as above, the therapeutic protein is an immunosuppressive therapeutic protein. In specific variations, an immunosuppressive therapeutic protein is selected from CTLA-4-Fc, TNFR-Fc, and an anti-TNFa antibody.

[0260] In certain embodiments of a system for polymer-mediated activation of engineered cells as above, the inducible promoter is a synNotch promoter. See, e.g., Morsut et al., supra, Roybal et al., supra,' Luo et al., supra, Moghimi et al., Nat. Commun. 12:511, 2021; Srivastava et al., Cancer Cell 35:489-503. e8, 2019; Choe et al., Set. Transl. Med. 13, 2021. In other embodiments, the inducible promoter comprises repeated transcriptional response elements (TREs) such as, for example, repeated NF AT TREs. See, e.g., Hooijberg et al., Blood 96:459-466, 2000; Zhang et al., Mol. Ther. 19:751-759, 2011. In still other embodiments, the inducible promoter is an inducible synthetic promoter (iSynPro) comprising multiple different TREs upstream from a minimal (core) promoter, such as described, e.g., in US Patent Application Publication No. 2020/0095573, incorporated by reference herein. A particularly suitable iSynPro promoter is an inducible promoter having the nucleotide sequence tcgaatgagtcacatcgatctccgccccctcttcgagggggcggggtcgaggaggaaaaactcgaatgagtcacatcgaccctttga tcttcgaggggactttccggggtggagcaagcgtgacaagtccacgtatgacccgaccgacgatatcgaagcctacgcgctgaacg ccagccccgatcgaccccgccccctcgatttccaagaaatcgaatgacatcatctttcgaatgacatcatctttcgaggggactttcctc gaacttccttcgaggggactttcctcgaggggactttcctcgaggaggaaaaactcgagtagagtctaga (SEQ ID NO:21).

[0261] In the present disclosure, engineered cells are prepared by introducing the inducible therapeutic protein transgene and a gene encoding the engineered receptor into cells using an expression vector. “Expression vector” means a nucleic acid molecule capable of transporting a nucleic acid molecule encoding the therapeutic protein (operatively linked to the inducible promoter) and/or a nucleic acid encoding the engineered receptor (operatively linked to a promoter for constitutive expression) into cells. The therapeutic protein transgene and gene encoding the engineered receptor may be contained within the same vector or may be present within separate vectors. An expression vector can be DNA or RNA in any form and of any origin, and various types of vectors are available. The vector can be a viral vector or a non-viral vector. Examples of viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and Sendai virus vectors. Among these, with a retrovirus vector, a lentivirus vector, and an adeno-associated virus vector, the gene of interest incorporated into the vector is integrated into the host chromosome and stable and long-term expression is expected. Each viral vector can be prepared according to conventional methods or by using commercially available kits for this purpose. Examples of non-viral vectors include plasmid vectors, liposome vectors, and positively charged liposome vectors (see, e.g., Feigner el al., Proc. Natl. Acad. Set. USA, 84:7413-7417, 1987), YAC vectors, and BAC vectors.

[0262] An expression vector comprises an expression unit (“expression cassette”) for expressing a therapeutic protein and/or engineered receptor gene. An expression cassette usually comprises a promoter, a nucleic acid segment encoding the therapeutic protein and/or a nucleic segment encoding the engineered receptor, and a poly A addition signal. As the therapeutic protein and engineered receptor genes are driven by different promoters (an inducible promoter for the therapeutic protein and a constitutive promoter for the engineered receptor), an expression vector containing both genes will contain two separate promoters, each operatively linked to the appropriate coding sequence. Examples of promoters that can be used in the engineered receptor expression cassette include CMV-IE (cytomegalovirus early gene-derived promoter), SV40ori, retrovirus LTRSRa, EFla, and 0-actin promoter. Examples of poly A addition signal sequences include a poly A addition sequence of SV40, a poly A addition sequence of a bovine growth hormone gene, and a poly A addition sequence of globulin. The therapeutic protein or engineered receptor coding sequence is usually connected to the 3’ end of its respective promoter directly or via another sequence so that the promoter regulates expression of the therapeutic protein or engineered receptor gene, and the poly A addition signal sequence is placed downstream of the therapeutic protein and/or engineered receptor gene(s). The engineered receptor gene is constitutively transcribed into messenger RNA (mRNA) from such an expression unit, and the engineered receptor is translated from the mRNA and presented on the engineered cell surface. Transcription of the therapeutic protein is triggered upon engagement of the engineered receptor with the target universal epitope and transmission of the activation signal from the intracellular signaling domain to the inducible promoter as discussed herein.

[0263] An expression cassette may also comprise a gene for detection of gene expression (e.g., a reporter gene, a cell or tissue-specific gene, or a selectable marker gene), an enhancer sequence for improving expression efficiency, or a WRPE sequence, for example. The gene for detection is used, e.g., for determining success or failure and efficiency of introduction of an expression vector, detecting expression or determining expression efficiency of the engineered receptor or therapeutic protein gene, or selecting or sorting cells expressing the engineered receptor gene. Examples of genes for detection or selection include the neo gene that confers resistance to neomycin, the npt gene (see Herrera-Estrella, EMBO J. 2:987-995, 1983) and the nptll gene (see Messing and Vierra, Gene 19:259-268, 1982) that confer resistance to kanamycin or other antibiotics, the hph gene that confers resistance to hygromycin (see Blochlinger and Diggelmann, Mol. Cell. Bio. 4:2929-2931, 1984), and the dhfr gene that confers resistance to methotrexate (see Bourouis and Jarry, EMBO J. 2: 1099- 1 104, 1983). Examples of marker genes include the luciferase gene (see Giacomin, Plant Sci. 116:59-72, 1996; Scikantha, J. Bad. 178:121, 1996), 0 -glucuronidase (GUS) gene, genes for fluorescent proteins such as GFP (see Gerdes and Kaether, FEBS Lett. 389:44-47, 1996) or variants thereof (e.g., EGFP, d2EGFP).

[0264] The expression vector prepared for gene transfer is introduced into cells by conventional methods. In the case of a viral vector, it is introduced into cells by viral infection. In the case of a non-viral vector such as a plasmid, conventional methods such as methods mediated by electroporation, liposome, or calcium phosphate can be used for introduction into cells, and the introduction is preferably carried out by electroporation.

[0265] In order to improve efficiency of integration into the host chromosome, it is preferable to carry out the gene transfer by a method mediated by a transposon. The transposon-mediated method is a non-viral gene transfer method, and it can integrate a gene of interest into the host chromosome by utilizing the mechanism by which an enzyme acting on the genome (transposase) and its specific recognition sequence cause gene translocation in combination. The transposon-mediated method can be, for example, the piggyBac transposon- mediated method. The piggyBac transposon-mediated method utilizes a transposon isolated from an insect (see Fraser et al., Insect Mol. Biol. 5: 141-51, 1996; Wilson et al., Mol. THER 15: 139-45, 2007) and it enables highly efficient integration into mammalian chromosomes. The use of the piggyBac transposon-mediated method for gene transfer is well known in the art (see, e.g., Nakazawa et al., J. Immunother. 32:826-836, 2009; Nakazawa et al., J. Immunother. 6:3-10, 2013). The transposon-mediated method for gene transfer is not limited to using piggyBac and can instead use another transposon such as, e.g., Sleeping Beauty (see Ivies et al., Cell 91 :501-510, 1997), Frog Prince (see Miskey et al., Nucleic Acids Res. 31 :6873-6881, 2003), Toll (see Koga et al., Mol. Gen. Genet. 249:400-5, 1995; Koga et al., J. Hum. Genet. 7:628-35, 2007), Tol2 (see Koga et al., Biotechnol. 4:6-11, 2002; Hamlet et al., Genesis 44:438-445, 2006; Choo et al., BMC Dev. Biol. 6:5, 2006).

[0266] The process of gene transfer by the transposon-mediated method can be a conventional process. For example, a piggyBac transposon-mediated method can be carried out by preparing a vector carrying a gene encoding the piggyBac transposase (a transposase plasmid) and a vector having a structure in which a gene expression cassette is sandwiched between piggyBac reverse repeat sequences (a transposon plasmid) and introducing these vectors into target cells by any of various methods such as electroporation, nucleofection, lipofection, or a calcium phosphate-mediated method.

[0267] As previously noted, a polymer displaying a universal epitope and a targeting moiety may have a naturally or synthetically produced polymer backbone. A universal epitope or targeting moiety may be part of a monomer from which one or more repeating units are derived during polymerization to produce the polymer. Alternatively, a universal epitope or targeting moiety may be bound to a repeating unit after polymerization via, e.g., conjugation to reactive groups on the polymer. Typically, a polymer is a “biocompatible polymer,” i.e., a polymer that is suitable for contact with bodily tissues and fluids because it does not cause an allergic reaction or other significant adverse side effects within a relevant dosage range. In some variations, a polymer displaying a universal epitope and a targeting moiety comprises a biopolymer such as, e.g., a polypeptide. For example, a polypeptide may be used in which both the universal epitope and the targeting moiety are peptides that are encoded in the polypeptide. In other variations, a polymer in accordance with the present disclosure comprises non-naturally occurring repeating units such as, for example, discussed further herein in the context of synthetic polymers.

[0268] In some variations, a polymer for use in accordance with the present disclosure has a molecular weight of from about 5 kDa to about 200 kDa. More typically, the polymer is from about 5 kDa to about 150 kDa, from about 10 kDa to about 100 kDa, or from about 15 kDa to about 100 kDa. In certain embodiments, the polymer is from about 20 kDa to about 80 kDa or from about 25 kDa to about 70 kDa. In other embodiments, the polymer is from about 20 kDa to about 60 kDa, from about 20 kDa to about 55 kDa, from about 20 kDa to about 50 kDa, from about 20 kDa to about 45 kDa, or from about 20 kDa to about 40 kDa. In still other embodiments, the polymer is from about 30 kDa to about 60 kDa, from about 35 kDa to about 60 kDa, from about 30 kDa to about 55 kDa, from about 35 kDa to about 55 kDa, or from about 40 kDa to about 50 kDa.

[0269] In some embodiments of a system as above, the universal epitope is pendant to a plurality of the repeating units of the polymer. In other, non-mutually exclusive embodiments, the targeting moiety is pendant to a plurality of the repeating units of the polymer. In some specific variations, from about 2% to about 15% or from about 2% to about 10% of polymer repeating units have the pendant universal epitope (e.g., about 2%, about 3%, about 4%, about 5%, about 8%, or about 10% of repeating units have the pendant universal epitope). In some non-mutually exclusive embodiments, the polymer comprises from about 2 to about 20, from about 3 to about 20, from about 3 to about 18, from about 3 to about 15, or from about 3 to about 10 repeating units having the pendant universal epitope.

[0270] In some variations of a system as above, the polymer is a synthetic polymer. Preferably, a synthetic polymer is produced by controlled polymerization. In one embodiment, the polymer is synthesized by living polymerization techniques such as, e.g., reversible addition-fragmentation chain transfer (RAFT) or Atom transfer radical polymerization (ATRP). Other methods of polymer synthesis that are known may be employed. Exemplary synthetic polymers suitable for use in accordance with the present systems and methods are disclosed in the studies described herein. Synthetic polymers amenable to use in accordance with the present disclosure are also described, e.g., in U.S. Patent No. 10, 231,993 to Pun et al., incorporated by reference herein.

[0271] In some embodiments of a synthetic polymer, the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units. Suitable hydrophilic repeating units include carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy- ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2- hydroxypropyl)methacrylamide (HPMA). In some embodiments, the synthetic polymer is a block copolymer (e.g., a block copolymer comprising a first block of repeating units having a pendant universal epitope and a second block of hydrophilic repeating units as above). In alternative embodiments, the synthetic polymer is a statistical copolymer (e.g., a statistical copolymer comprising a first plurality of repeating units having a pendant universal epitope and a second plurality of repeating units that are hydrophilic repeating units as above).

[0272] In particular embodiments of a system as above, the engineered cell is a CAR immune cell (e.g., a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell) and the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment. Accordingly, in some such variations, the system is a system for polymer-mediated activation of a CAR immune cell generally including

(i) a universal chimeric antigen receptor (CAR) epitope, and

(b) a CAR immune cell comprising

[0273] More particular embodiments of a system for polymer-mediated activation of a CAR immune cell as above include, e.g., various embodiments as previously described, including described embodiments of universal epitopes, targeting moi eties (e.g., ECM-binding peptides), extracellular binding domains, CAR intracellular signaling and costimulatory domains, inducible promoters (e.g., iSynPro promoters such as, for example, the promoter of SEQ ID NO:21), therapeutic proteins, and polymers. Particularly suitable therapeutic proteins include (i) a second CAR that specifically recognizes a tumor-associated antigen expressed by a solid tumor cell (e.g., a bispecific CAR that specifically recognizes two different tumor- associated antigens), (ii) an antibody that specifically recognizes a tumor-associated antigen expressed by a solid tumor cell (e.g., a bispecific antibody such as, for example, a bispecific T cell engager), (iii) an immune checkpoint inhibitor (e.g., anti-CTLA-4, anti-PD-l/PD-Ll, or a combination thereof), and (iv) an immunostimulatory cytokine (e.g., an immunostimulatory cytokine such as, for example, IL-2, IFN-a, IL-12, IL-15, or IL-21). In some variations, an encoded therapeutic protein is a second CAR that specifically recognizes a second universal epitope (which may be present, e.g., on a second polymer or intermediate adaptor molecule).

[0274] Systems comprising an engineered cell and a cognate polymer as described herein may be used to deliver a therapeutic protein to a tissue or cell of interest in a subject. Such methods are particularly useful, e.g., for delivering a therapeutic protein for the treatment of various diseases or disorders by targeting and concentrating therapeutic protein delivery to sites of disease pathology while minimizing off-target effects. In certain variations, the systems of the present disclosure are used to deliver therapeutic proteins to provide immune protection against cancer or infections by pathogens (e.g., viral or bacterial infections). In some variations, the systems of the present disclosure may be used to deliver therapeutic proteins that down-modulate an immune or inflammatory response such as, e.g., for treatment of autoimmune disease, allergic diseases, or other immunological or proinflammatory conditions.

[0275] Accordingly, in another aspect, the present disclosure provides a method for expressing a therapeutic protein at the site of a tissue or cell of interest in a subject using a system as described herein. The method generally includes administering to a subject an effective regimen of

(i) a universal epitope, and

(b) an engineered cell comprising

(B) an intracellular signaling domain derived from a cell surface receptor, and

[0276] More particular embodiments of a method for expressing a therapeutic protein at the site of a tissue or cell of interest as above include, e.g., those utilizing any of various embodiments of a system as previously described, including described embodiments of universal epitopes, targeting moi eties (e.g, ECM-binding peptides), engineered receptors (e.g., CARs or synNotch receptors), engineered cells (e.g., CAR immune cells), extracellular binding domains, intracellular signaling domains (e.g., CAR signaling and costimulatory domains), inducible promoters (e.g., iSynPro promoters such as, for example, the promoter of SEQ ID NO:21), therapeutic proteins, and polymers. In some embodiments, the polymer is administered before the engineered cell. In other embodiments, the engineered cell is administered before the polymer.

[0277] In certain embodiments, a method for expressing a therapeutic protein is a method for expressing an anticancer therapeutic protein at the site of a solid tumor cancer. Accordingly, in a related aspect, the present disclosure provides a method for treating a solid tumor cancer using a system as described herein. The method generally includes administering to a subject having the solid tumor cancer an effective regimen of

(i) a universal chimeric antigen receptor (CAR) epitope, and

(b) a CAR immune cell comprising

[0278] More particular embodiments of a method for treating a solid tumor cancer using a system as described herein as above include, e.g., those utilizing any of various embodiments of a system as previously described wherein (i) the engineered cell is a CAR immune cell, (ii) the targeting moiety is an ECM component of the tumor microenvironment, and (iii) the therapeutic protein has anticancer therapeutic efficacy. Such embodiments include previously described embodiments of universal epitopes, ECM components for targeting, ECM-binding peptides, extracellular binding domains, CAR signaling and costimulatory domains, inducible promoters (e.g., iSynPro promoters such as, for example, the promoter of SEQ ID NO:21), and polymers. In some embodiments, the polymer is administered before the CAR immune cell. In other embodiments, the CAR immune cell is administered before the polymer. Exemplary solid tumor cancers amenable to treatment according to the disclosed method include breast cancer, colorectal cancer, hepatocellular carcinoma, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, basal cell carcinoma, cutaneous squamous cell carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, cervical cancer, endometrial carcinoma, esophageal carcinoma, gastric carcinoma, and Merkel cell carcinoma.

[0279] In certain variations of a method for treating a solid tumor cancer as above, the encoded anticancer therapeutic protein is selected from (i) a second CAR that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells, (ii) an antibody that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells, (iii) an immune checkpoint inhibitor, and (iv) an immunostimulatory cytokine. In some embodiments wherein the encoded therapeutic protein is a second CAR that specifically recognizes a tumor- associated antigen, the second CAR is a bispecific CAR that specifically recognizes two different tumor-associated antigens expressed by the solid tumor cells. In some embodiments wherein the encoded therapeutic protein is an antibody that specifically recognizes a tumor- associated antigen, the antibody is a bispecific antibody and/or a single chain antibody. In some embodiments comprising a bispecific antibody, the antibody is a bispecific T cell engager. In other embodiments wherein the therapeutic protein an immune checkpoint inhibitor, the immune checkpoint inhibitor is anti-CTLA-4, anti-PD-l/PD-Ll, or a combination thereof. In some embodiments wherein the encoded therapeutic protein is an immunostimulatory cytokine, the immunostimulatory cytokine is selected from the group consisting of interleukin-2 (IL -2), interleukin-4 (IL-4), interferon-alpha (IFN-a), interleukin- 12 (IL-12), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

[0280] For therapeutic use, a population of engineered cells and a polymer as described herein are administered as an effective regimen appropriately determined according to factors such as age, body weight, and symptoms of the subject. The subject of the present disclosure is typically a human (e.g., a cancer patient such as in the case of a method for treating a cancer), but in some embodiments a patient is a nonhuman mammal. The engineered cells and polymer as described herein are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought.

[0281] The engineered cells (e.g., CAR immune cells) of the present disclosure can be administered, for example, from about 1 x 10⁴ cells to about 1 x io¹⁰ cells at a time. In addition to the engineered cells to be administered to a subject, a composition comprising the engineered cells may further include a component such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting cells, an antibiotic for the purpose of preventing contamination by bacteria, or any of various components for the purpose of activating, proliferating or inducing differentiation of cells (such as vitamins, cytokines, growth factors, steroids, and the like). The composition can be prepared by conventional methods.

[0282] For administration of a polymer of the present disclosure, a dosage typically ranges from about 0.1 pg to 100 mg/kg or 1 pg/kg to about 50 mg/kg, and more usually 10 pg to 5 mg/kg of the subject’s body weight. In more specific embodiments, an effective amount of the agent is between about 1 pg/kg and about 20 mg/kg, between about 10 pg/kg and about 10 mg/kg, or between about 0.1 mg/kg and about 5 mg/kg. Dosages within this range can be achieved by single or multiple administrations.

[0283] A polymer for administration in accordance with the present disclosure is formulated as a pharmaceutical composition. A pharmaceutical composition comprising a polymer as described herein can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See, e.g., Gennaro (ed.), Remington ’s Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995). Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent polymer loss on vial surfaces, etc.

[0284] The administration route of the engineered cells and polymer is not particularly limited to, but can be, for example, intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The engineered cells and polymer may be administered systemically or locally, and the local administration includes direct injection into a target tissue or organ. The administration schedule is appropriately determined according to factors such as age, body weight, and symptoms of the subject, and may be a single administration or a continuous or periodic multiple administrations. The engineered cells and polymer may be administered at either the same site or at different sites, by the same or different administration routes, and according to the same or different administration schedules. For example, the polymer may be administered either before or after administration of the engineered cells. In some alternative variations, the polymer and engineered cells are administered concurrently, e.g., at different sites. In certain non-mutually exclusive embodiments, both the engineered cells are administered intravenously. In other non-mutually exclusive embodiments, the polymer is administered according to a repeat dosing schedule following a single bolus delivery of the engineered cells, e.g., to maintain or re-stimulate activation of the engineered cells at a target tissue site over an extended period.

[0285] Determination of an effective treatment regimen is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and regimens that significantly reduce the occurrence or severity of the subject disease or disorder in model subjects. An effective regimen of the system components varies depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, whether treatment is prophylactic or therapeutic, as well as the specific activity of the system itself and its ability to elicit the desired response in the individual. Typically, treatment regimens are adjusted to provide an optimum therapeutic response, i.e., to optimize safety and efficacy. Accordingly, a therapeutically or prophylactically effective regimen is also one in which any undesired collateral effects are outweighed by beneficial effects.

[0286] For treatment of solid tumor cancers, CAR immune cell/polymer systems in accordance with the present disclosure can be evaluated for anti-tumor activity in well-known animal tumor models. Exemplary animal models include syngeneic tumor-bearing mice models of cancer using murine CAR T cells (e.g., mice bearing 4T1 breast cancer cells such as used by Zhang etal., Cancer Research 78:3718, 2018, or AE17om mesothelioma cells such as used by Klampatsac/ c//., Molecular Therapy 18:360, 2020) and immunocompromised mice models or humanized mice using xenograft human tumors (as reviewed, e.g., in Mhaidly and Verhoeyen, Cancers 12: 1915, 2020; Siegler and Wang, Human Gene Therapy 29, 2018).

[0287] The disclosure is further illustrated by the following non-limiting examples.

Example 1; Intratumoral Activation of Fluorescein-Specific CAR T Cells Following Administration of a Synthetic Fibrin-homing Polymer

[0288] This example describes an exemplary system for polymer-mediated activation of an engineered cell comprising a transgene with an inducible promoter, wherein the inducible promoter is driven by an activation signal from an engineered receptor expressed by the cell. The transgene encodes an mCherry:ffluc reporter protein and is shown herein to be expressed at the site of a target tissue in an inducible-promoter-restricted manner only upon engagement of the engineered receptor with its ligand on the polymer. The mCherry:ffluc protein in this example serves as a surrogate for any therapeutic protein and therefore provides a proof-of- concept demonstration for engineered cell therapies with an inducible-promoter-restricted therapeutic payload in accordance with the present disclosure.

Introduction

[0289] Universal CARs are a class of receptors that may offer a potential solution to both antigen escape and off-site toxicities. See Minutolo etal., Front. Oncol. 9:176, 2019. T cells featuring universal CARs can engage tumors exclusively via synthetic bifunctional intermediate adaptor molecules, which present both CAR epitope and tumor targeting moieties. The dependency of the CAR on these bridging molecules for antigen recognition and immune synapse formation allows for highly regulated, multivalent CAR T cell effector function. For example, multiple tumor antigens can be simultaneously targeted with a single CAR T cell by creating a panel of intermediate adaptors with a constant CAR epitope but different tumor targeting moieties. The strength of the T cell response can then be modulated by adjusting the dose and frequency of intermediate adaptors given to the patient. The specificity of this response can be refined further by implementing an IF-THEN gate on effector function. IF-THEN gates enable conditional expression of a therapeutic agent or CAR via an inducible promoter. It is therefore possible to put a tumor-targeting CAR under the regulation of an inducible promoter and define an “IF” condition that creates a spatial distinction between tumors and surrounding tissue. One way to accomplish this is by taking advantage of irregular coagulation patterns common in malignant tissue. Fibrin clots are hallmark components of wounds and tissue regeneration that are rarely observed under normal conditions. See Obonai et al., Sci. Rep. 6:23613, 2016. Since solid tumors are destructive to surrounding tissue and require continuously expanding vasculature to grow, they are often characterized by significant fibrin deposits. See Dvorak et al. , N. Engl. J. Med. 315: 1650- 1659 (1986).

[0290] This study describes an exemplary framework for IF-THEN gating CAR T cell function called “Zu situ Mobilization: Polymer Activated Cell Therapies” (IMPACT) (see Figure 1). In this system, PolySTAT, a fibrin-binding polymer see Lamm et al., ACS Biomaterials Sci. Eng. 6:7011-7020, 2020; Chan et al., Sci. Transl. Med. 7:277ra29-277ra29, 2015) was adapted to bind within the tumor microenvironment (TME) and provide an “IF” condition to logic-gated CAR T cells. PolySTAT was modified to display multiple fluorescein (FL) tags along its backbone for recognition by a constitutively expressed anti-FL universal CAR (“cCAR”) on T cells. See Kim et al., J. Am. Chem. Soc. 137:2832-2835, 2015; see also A Phase I Feasibility And Safety Study of Fluorescein-Specific (FITC-E2) CAR T Cells In Combination With Parenterally Administered Folate-Fluorescein (UB-TT170) For Osteogenic Sarcoma, ClinicalTrials.gov website, Study ID NCT05312411, 2022. Upon engagement with its epitope, FL CAR signaling activates inducible synthetic promoter (iSynPro), which is a synthetic promoter responsive to TCR or CAR signaling. See U.S. Patent Application Publication No. 2020/0095573. iSynPro then drives expression of an mCherry:ffluc fusion protein (“iCherry”) in an anatomically-restricted manner. These cCAR T cells are a proof-of- concept platform to model engineered cell therapies with a synthetic-promoter-restricted therapeutic payload, such as, e.g., a bispecific T cell engager (BiTE; see Staerz et al., Nature 314:628-631, 1985) or another CAR that can target a (tumor-associated antigen) TAA or an intermediate adaptor. Materials and Methods

Material for Polymer synthesis

[0291] Fluorescein O-methacrylate (FMA), fluorescein 5(6)-isothiocyanate

(BioReagent, mix of isomers, >90% by HPLC), 2-aminoethyl methacrylate hydrochloride (AEMA), 2,2'-Azobis(2-methylpropionitrile) (AIBN), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CCC), 2-hydroxyethyl methacrylate (HEMA), and all other reagents were purchased from Sigma-Aldrich (Saint Louis, MO) unless noted otherwise. N-hydroxysuccinimide methacrylate (NHSMA) was purchased from TCI America (Portland, OR). Glycerol monomethacrylate (GmMA) was purchased from Polysciences, Inc. (Warrington, PA). The fibrin binding peptide (FBP; Sequence: Ac-Y(DGl)C(HPr)(Y(3-Cl))GLCYIQGK-NH2; SEQ ID NO:4) (Kolodziej et al., Bioconjug. Chem. 23:548-556, 2012), developed by the Caravan group, as well as the scrambled peptide (Ac-YICGQ(DGl)AC(HPr)LYGK-NH2; SEQ ID NO:22) were both purchased from two suppliers, GL Biochem (Shanghai, China) and Elim Biopharm (Hayward, California), as custom orders. Human fibrinogen, thrombin, and plasmin were purchased from Enzyme Research Laboratories (South Bend, IN). Human Fibrinogen Purified FL Labeled (4- 8 FL/fibrinogen) was purchased from Molecular Innovations (Novi, MI).

Cloning of the PiggyBac Construct

[0292] All oligonucleotides used were synthesized by Integrated DNA Technologies. All DNA fragments were available in-house. Different sections of donor plasmids, including a piggyBac transposon vector (Aldevron), the anti -FL (E2-mut2) CAR, iSynPro promoter, and mCherry:ffluc fusion protein were digested using Nhel, BlpI, Notl, Sall, Nrul, and PacI restriction enzymes (NEB). Digested DNA fragments were gel purified with a ZymoClean Gel DNA Recovery Kit (Zymo Research) and PCR amplified with Q5® High-Fidelity DNA Polymerase master mix (NEB). PCR products were again gel purified with a ZymoClean Gel DNA Recovery Kit and ligated together via Gibson assembly using Takara’ s In-Fusion® Snap Assembly Master Mix. Stellar™ chemically competent E. coli (Takara) were transformed with the Gibson assembly products, and kanamycin-selected colonies were screened by PCR for correct insert lengths using SapphireAmp® Fast PCR Master Mix (Takara). Correct DNA sequences at ligation sites were verified by Sangar sequencing (Genewiz) of miniprep DNA (Qiagen). Final colonies were then selected and grown to prepare transfection-grade plasmid DNA via maxiprep (Macherey -Nagel).

T Cell Isolations

[0293] For all donor cells used in this study, Leukocyte Reduction System (LRS) cones were obtained from Bloodworks Northwest. CD4+ and CD8+ T cells were isolated with EasySep HLA CD4 and CD8 Chimerism Kits and a RoboSep™-S Automated Cell Separation Instrument (STEMCELL Technologies). PBMCs from negative fractions were then isolated using SepMate™ PBMC Isolation Tubes (STEMCELL Technologies). Aliquots of CD4⁺ and CD8⁺ T cells were set aside and stained with anti-CD45RO APC (BioLegend), anti-CD62L PE (BioLegend), anti-CD4 FITC (BioLegend), and anti-CD8 BUV395 (BD Horizon). After the stain and subsequent washes, cells were fixed in 0.5% paraformaldehyde and kept in the dark at 4°C until running on a LSRII Fortessa (BD Biosciences). Compensation was performed using tubes of UltraComp eBeads Compensation Beads (ThermoFisher Scientific) individually stained for each specific fluorophore used, and the compensation matrix was calculated using Flowlo Software (TreeStar). Flowlo software was also used for data analysis.

7 Cell Manufacturing

[0294] Freshly isolated CD8+ T cells were cultured in RPMI (Gibco) with a final concentration of 10% heat-inactivated and gamma irradiated FBS (VWR) and 2mmol/L L- Glutamine (ThermoFisher Scientific) (Complete RPMI) plus 4.6ng/mL TL2 (STEMCELL Technologies) and 0.5ng/mL IL15 (Miltenyi). Cells were stimulated with 25pL /mL ImmunoCult™ Human CD3/CD28 T cell Activator for 72 hours. After 72 hours, cells were centrifuged and prepared for electroporation (EP) using a 4D-Nucleofector™ X Kit (Lonza). Each well, except mock cells, received lOnM DNA and 0.528pL of Piggybac transposon RNA. After EP, cells were immediately added to warm cytokine supplemented complete RPMI following electroporation and incubated for 3 days. Methotrexate (Medline) was then added to each CAR T cell well, and half medium changes were performed every 2-3 days. 21 days after EP, cells stained with anti-CD19 SB600 (ThermoFisher Scientific) to assess CAR positivity via flow cytometry. Cell Line Culturing

[0295] K562s (an erythroleukemia cell line) were obtained from the European Collection of Cell Cultures through Sigma-Aldrich. K562 OKT3 cells were made by lentiviral transduction of an OKT3scFv-CD4tm-T2A-Her2tG_epHIV7.2 vector into the K562 parental cell line, thereby leading to expression of an anti-CD3 agonist OKT3scFv. K562 cell lines were cultured in RPMI (Gibco) with a final concentration of 10% heat-inactivated/gamma irradiated FBS (Seradigm) and 2mmol/L L-Glutamine (ThermoFisher Scientific) in 5% CO2 at 37°C. BT-20 cells were obtained from ATCC and cultured in DMEM (Gibco) with final concentration of 10% heat-inactivated/gamma irradiated FBS (Seradigm) and 2mmol/L L- Glutamine (ThermoFisher Scientific) in 5% CO2 at 37°C. All lines were tested for mycoplasma contamination.

Synthesis of 2-Propenoic acid, 2-methyl-, 2-[[[(3',6'-dihydroxy-3-oxospiro

[ isobenzofur an- 1(3H), 9 ’-[ 9H]xanthe ] -5-yl) amino Jthioxomethyl ] amino ] ethyl ester (AEMA-FL)

[0296] Fluorescein 5(6)-isothiocyanate (1 g/2.58 mmol) and 1.1 equivalents of 2- aminoethyl methacrylate hydrochloride (470 mgs/2.84 mmol) were added to a round bottom flask with a stir bar. DMSO (25.8 mL/0.1 M) was added. Once the constituents were dissolved (pale yellow solution), 2.6 equivalents of DIPEA (1.29 mL) were added dropwise (turned to deep orange color). The mixture was stirred at RT overnight. The solution was precipitated one time in cold 6% HC1 solution (1 Ox volume of reaction) constantly swirled over ice. The orange precipitate was filtered off with a glass frit. The precipitate was collected into a glass vial then lyophilized overnight. The solid, orange monomer was stored at -20 °C. Yield was > 90%. The monomer was pure by ¹ H nuclear magnetic resonance (NMR) spectroscopy on a Bruker AV 300 and TLC.

Synthesis of Statistical Copolymer Backbones

[0297] pHEMA-co-FMA-co-NHSMA, pHEMA-co-AEMA-FL-co- NHSMA, and pGmMA-co-AEMA-FL-co-NHSMA were synthesized via reversible additionfragmentation chain transfer (RAFT) polymerization as described previously. See Chan el al., Sci. Transl. Med. 7:277ra29-277ra29, 2015. Briefly, comonomers HEMA or GmMA were combined with the FL comonomer (FMA or AEMA-FL) and NHSMA at different ratios to achieve desired FL content. The comonomers were combined with CCC and AIBN at 200: 1 :0.333 ratio in dimethylacetamide at a monomer concentration of 0.6 M. This mixture reacted for 20 h at 70 °C. pHEMA copolymers were precipitated in diethyl ether, redissolved in dimethylacetamide, and precipitated again in diethyl ether. pGmMA copolymers were precipitated in diethyl ether followed by dissolution in dimethylsulfoxide and a second precipitation in 50-50 acetone/diethyl ether. Precipitated polymer was collected by centrifugation at 7197 x g. Dithiobenzoate groups were removed via an end-capping reaction with 20x molar excess AIBN at 70 °C for 12 hours.

Synthesis of Block Copolymer Backbones

[0298] First, macroCTAs (activating blocks) were synthesized via RAFT. A CTA:I ratio of 5: 1, target DP of 30, and monomer concentration of 0.6M were used for all the reactions. The ratio of GmMA to AEMA-FL and polymerization time were changed to hit a desired number of FL molecules in the MacroCTA. MacroCTAs were precipitated in diethyl ether followed by dissolution in dimethylsulfoxide and a second precipitation in 50- 50 acetone/diethyl ether. Precipitated polymer was collected by centrifugation at 7197 x g. Dissolution in DMSO followed by precipitation in 50-50 actetone/di ethyl ether was repeated 3-5 times to remove residual monomer (monitored by

nuclear magnetic resonance spectroscopy). The macroCTAs were chain extended with GmMA and NHSMA at a GmMA:NHSMA:MacroCTA:AIBN ratio of 160:40: 1:0.333 in dimethylacetamide with a monomer concentration of 0.6 M. This mixture reacted for 20 h at 70 °C. Copolymers were precipitated in diethyl ether followed by dissolution in dimethyl sulfoxide and a second precipitation in 50-50 acetone/diethyl ether. Precipitated polymer was collected by centrifugation at 7197 x g. Dithiobenzoate groups were removed via an end-capping reaction with 20x molar excess AIBN at 70 °C for 12 hours.

Synthesis of Poly STAT via Conjugation

[0299] Poly STAT was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as described previously. See Chan et al., supra, Lamm etal., Biomaterials 132:96-104, 2017. Backbone polymers synthesized as described above were conjugated to FBP via reaction of the C-terminal lysine in the peptide under organic basic conditions in DMSO at a varying ratios of peptide:NHS with N.N-diisopropylethylamine added at a 5: 1 ratio base:peptide (see Yanjarappa et al., Biomacromolecules 7:1665-1670, 2006) for 24 h at 50 °C, after which unreacted NHSMA groups were capped with lOx molar ratio of l-amino-2-propanol. Peptide-polymer conjugates were purified by extensive dialysis as follows. First, the product was dialyzed against phosphate-buffered saline (PBS) for 24 h (3 buffer changes, 4 L of buffer) during which a precipitate formed. Contents of the dialysis bag were collected and centrifuged at 4500 x g for 8 min to remove insoluble material; the supernatant was collected and moved to a fresh dialysis bag. Dialysis continued for 24 h (3 buffer changes), followed by -pH 8 water (NaOH added) for 48 h (6 dialysate changes) to remove PBS salts. To synthesize control polymers for UV-Vis characterization of FL content, the same exact procedure was followed, however no peptide was added for conjugation and all NHSMA groups were capped with 1 -amino-2-propanol. Purification was identical.

Polymer Characterization

[0300] Polymers were characterized via GPC in dimethylformamide with static light scattering and refractive index detectors (MiniDawn Treos and OptilabTRex, respectively, both from Wyatt Technology, Santa Barbara, CA) to determine molecular weight and dispersity index (PDI). 'H nuclear magnetic resonance (NMR) spectroscopy on a Bruker AV 300 was utilized to determine conversion of the polymer prior to purification, and composition after purification. Conversion of the FL comonomers (FMA and AEMA-FL) were monitored during polymerization to estimate final FL content. Ultraviolet-visible spectroscopy (UV- Vis) was used to determine FL content in the final control polymers.

ROTEM Characterization of FL-PolySTATs from Various Synthesis Strategies

[0301] ROTEM whole blood hemostasis analyzer (ROTEM, Instrumentation Laboratory, Bedford, MA, USA) was used to confirm activity of different PolySTAT formulations as previously described. See Lamm et al. , ACS Biomaterials Sci Eng 6:7011-7020, 2020; Chan et al., ACS Biomater. Sci. Eng. 2:403-408, 2016. Briefly, 300 pL of a clotting solution with final concentrations in the ROTEM were 1.5 mg/mL fibrinogen, 0.5-1 TU/mL thrombin, 2-4 pg/mL plasmin, 0.1 mmol/L CaCE, and 5 pmol/L PolySTAT at pH 7.4. Measured parameters in ROTEM included: (i) the clotting time (CT), measured as the time between reagent addition to clot formation; (ii) a-angle, which reflects the rate of clot formation, (iii) the maximum clot firmness (MCF), the highest strength observed for the clot, (iv) the lysis index-30 minutes (LI-30), the percentage of MCF retained 30 minutes after initiation of clot formation, and (v) maximum lysis (ML), the percentage of clot strength lost compared to the MCF at the end of analysis.

Poly STAT Gel Manufacturing

[0302] To make PolySTAT gels, we first made compounded HEPES buffer (44nM HEPES (Gibco), 2nM CaCh, and 140mM NaCl in PBS pH 7.4). Compounded HEPES buffer (145 pL) was then mixed with 10 pL of 20 mg/mL CaCb and 2.5 pL of 80 lU/mL thrombin, thereby making the “activating mixture.” The activating mixture was then mixed with 5 pL of PolySTAT in a 48-well plate (for 5uM PolySTAT gels, the PolySTAT stock would be at 200uM). Last, 40 pL of 10 mg/mL fibrinogen was carefully mixed in to avoid forming bubbles. Completed gels incubated at 37 °C for 1 hour followed by a 5-minute incubation at - 20 °C to bring the plate to room temperature.

PolySTAT-ELISA protocol

[0303] To analyze anti-FL antibody binding to PolySTAT, PolySTAT-loaded fibrin gels were washed with PBS and blocked with 5% BSA (ENZO life sciences) for 1 hour at room temperature. The BSA was then aspirated off the gels and 1 pg/mL of biotinylated anti- Fluorescein antibody (abeam) was added onto the gels in 5% BSA PBS solution for 20 minutes. Gels were then washed five times with PBS + 0.05% Tween20. For each wash, gel plates were placed on a shaker at 700 RPM for 2 minutes. After the washes, streptavidin-HRP in 200uL was added to each well and incubated for 20 minutes. Gels were then washed three times with PBS + 0.05% Tween20 and three times with PBS. A QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit (ThermoFisher Scientific) was then used for the ADHP/HRP enzymatic reaction. After a 15-minute reaction, reactions were quenched with the provided reagent and plates were imaged on a plate reader.

In Vitro Cell Activation Experiments: Cells

[0304] CD69 and mCherry expression were examined after stimulation of 500,000 mock or cCARiCherry CAR T cells with a 1 : 1 E:T co-incubation with K562 Parental, K562 OKT3, or FL-PLE-labeled K562 Parental cells. T cells and target cells were co-incubated in 200pL of cytokine-free compounded media at 37°C and 5% CO2 for 24 hours. Cells were then harvested and stained with live/dead FVS780 (BD Horizon), anti-CD19 BV421 (BioLegend), and anti-CD69 APC (BioLegend). Cells were then fixed with 0.5% paraformaldehyde and kept in the dark at 4°C until running on a LSRII Fortessa (BD Biosciences). FlowJo software was used for data analysis. Compensation was performed using UltraComp eBeads Compensation Beads (ThermoFisher Scientific) and FlowJo Software (TreeStar) to analyze data.

In Vitro Cell Activation Experiments: Gels

[0305] CD69 and mCherry expression were examined after stimulation of cCARiCherry CAR T cells with different polymer-loaded gels. Statistical and block AEMA- FL-PolySTAT (5pM) and associated PolySCRM-loaded gels were manufactured as described above. One million mock or cCARiCherry CAR T cells were added to designated wells in 200pL of cytokine-absent compounded media. 5% FL-fibrinogen gels, 1 : 1 E:T FL-PLE- labeled K562 cells, and 100X Cell Stimulation Cocktail (ThermoFisher Scientific) were used as positive controls. Plates were then placed in an IncuCyte® Live-Cell Analysis System (Essen Bioscience) at 37 °C and 5% CO2, and images were taken every 2 hours for 20 hours. After the incubation, samples were harvested and stained with live/dead FVS780 (BD Horizon), anti-CD19 BV421 (BioLegend), and anti-CD69 APC (BioLegend). Cells were then fixed and analyzed on a LSRII Fortessa as described.

Poly STAT In Vivo Tumor Localization Study

[0306] Five million BT-20 breast cancer cells (ATCC) were engrafted subcutaneously in six NSG mice. Tumors were allowed to grow for 4 weeks until tumors were ~80mm³. At this point, PBS or 10 mg/kg 2% statistical AEMA-FL PolySTAT or AEMA-FL-PolySCRM were injected IV into 2 mice/group. After 24 hours, mice were euthanized and immediately sequentially perfused with PBS and a 10% formalin solution. Tumors were excised and frozen in OTC. Frozen tumors were then cut into 10 pm thick slices using a cryotome and placed on clean microscope slides. Tumor slices were stained with a primary anti-fibrinogen antibody (abeam) and secondary AF647 antibody (Abeam) and an anti-FITC AF488 antibody (Jackson ImmunoResearch). Slices were imaged using a confocal microscope (Nikon).

In Vivo Cell Activation Study

[0307] Five million BT-20 breast cancer cells (ATCC) were engrafted subcutaneously in six NSG mice. Tumors were allowed to grow for 4 weeks until tumors were ~80mm³. At this point, 10 million mock or cCARiCherry T cells were intravenously administered. After 48 hours, PBS or 10 mg/kg 2% statistical AEMA-FL PolySTAT or AEMA-FL-PolySCRM were injected IV into 4 mice per condition. Mice were then administered 4.29mg of D- luciferin (Perkin Elmer) and imaged every 24 hours for 8 days with an IVIS imaging machine (Xenogen). Flux was quantified using Perkin Elmer’s Living Image® software. A one-way ANOVA test with repeated measures between the cCARiCherry|PolySTAT, cCARiCherry|PolySCRM, and cCARiCherry|PBS groups was performed, followed by Tukey’s HSD post-hoc analysis to compare each group with one another.

Image J Analyses

[0308] Confocal images were imported into Fiji (Image!) and split into individual RGB images. Red (fibrinogen) and green (PolySTAT) sub images were then converted to masks to produce pixel binary images for each color (signal or no signal). Masked images were imported into the JACoP plugin in Fiji to conduct distance-based colocalization analyses and generate a color overlap image. Each colocalization analysis produced an overlap percentage. Mean and standard deviation for overlap percentages were then calculated in Microsoft Excel.

Statistical Analyses

[0309] Statistical significance for PS-ELISA was performed using GraphPad software. A one-way ANOVA was performed on the groups broadly. Once population -wide significance was determined, a Tukey’s posttest was conducted to compare between groups. Data presented are mean values +/- SD. * ?<0.01, **/?<0.001, *** ?<0.0001. Statistical significance for in vivo studies was calculated using GraphPad software. For the experiment in our main figure, we performed a one-way ANOVA with a Tukey’s posttest to compare each group. Data presented are mean values +/- SD. **** =/?<0.0001, *** =/?<0.001, ** =/?<0.01, * =/2<0.05. For the experiment included in the supplemental work, we performed an unpaired one-tailed T test with a Welch’s correction for unequal variance. Data presented are mean values +/- SD, * = ><0.05. Results

Design of a PiggyBac Nanoplasmid Vector For Constitutive Expression of a Fluorescein-Specific CAR and Activation-Dependent Transcription of a mCherry ffLuc Reporter

[0310] IF-THEN-gated CAR T cells were designed that recognize an antigen via a constitutively-expressed CAR and subsequently induce expression of a reporter mCherry:ffluc protein (cCARiCherry) when stimulated (see FIG. 1). A plasmid was constructed that contains the inducible synthetic promoter, iSynPro, and the human eukaryotic translation elongation factor la (EFla) promoter, which constitutively drives high transgene expression. See Wang etal.,J. Cell. Mol. Med. 21 :3044-3054, 2017. Inserted under EFla control was an anti -FL(E2- mut2) CAR with CD19t expression marker and methotrexate (MTX)-resistant double mutant dihydrofolate reductase (DHFRdm) separated by 2A ribosomal skip sequences. See Jonnalagadda et al., Gene Ther. 20:853-860, 2013. Inserted under iSynPro regulation was an mCherry:ffluc fusion protein (see FIG. 2A), which allows for in vitro validation of cell activation via mCherry and in vivo validation of cell activation via ffluc. The iSynPro promoter stringently restricts expression of the mCherry:ffluc fusion protein until the CAR (or TCR) engages its ligand. Once the CAR-ligand interaction is engaged, the cells transiently transcribe the mCherry:ffluc transgene (see FIG. 2B). These components were all cloned into a piggyBac transposon vector (see Manuri et al., Hum. Gene Ther. 21 :427-437, 2010; Nakazawa et al., J. Immunother. 32:826-836, 2009; Nakazawa et al. , Mol. Ther. 19:2133-2143, 2011; Bishop et al., Mol. Ther. 26: 1883-1895, 2018) for electroporation (EP) into CD8⁺ primary human T cells. Following EP, CAR⁺ populations were selected with administration of 50 nM methotrexate for 18 days. At the end of the initial 21 -day cell production, cells underwent a rapid cell expansion protocol (REP). On the last day of REP, cell staining for CD19t revealed a 77.8% CAR⁺ population of cells when compared to untransduced, donor-matched negative control (mock) cells (see FIG. 2C). Once CAR expression was validated, it was confirmed that iSynPro was responsive to anti-FL CAR signaling. cCARiCherry T cells were incubated with the following at a 1 : 1 effector:target (E:T) ratio for 24 hours and then assessed for CD69 expression (an early T cell activation marker) (see Arva & Andersson, Scand. J. Immunol. 49:237-243, 1999) and mCherry expression: K562 OKT3 cells (positive control), which stimulate the T cells via endogenous TCRs, K562 parental (K562 P) cells (negative control), and K562 P cells labeled with FL-PLE (see FIG. 2D). T cells stimulated with both K562 0KT3 and K562 FL-PLE cells expressed mCherry and upregulated CD69; interestingly, mCherry expression was dependent on CAR expression (CD19t⁺ cells; see FIGs. 2E-2G), further confirming IF-THEN controlled gene expression. These data confirmed that the dual promoter system was functional and ready to be tested with PolySTAT.

Fluorescein Conjugation Chemistry, Polymer Composition and Polymer Structure Influence the Efficiency of cCARiCherry T Cell Activation

[0311] The PolySTAT platform was engineered to enhance the presentation of fluorescein (FL) and recognition by cCARiCherry T cells. Commercially available, fluorescein O-methacrylate (FMA) was co-polymerized with 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid N-hydroxysuccinimide ester (NHSMA) to create a 2% FL statistical copolymer backbone, followed by conjugation of a fibrin-binding peptide (FBP) to generate FMA-PolySTAT (see FIG. 6A). Fibrin gels containing escalating doses of FMA- PolySTAT or FMA-PolySCRM (a non-fibrin binding, scrambled peptide negative control for PolySTAT) were then constructed in 48-well tissue culture plates using previously-described materials and concentrations (see FIG. 6B). See Murphy & Leach, BMC Res. Notes 5:423, 2012. cCARiCherry T cells or mock cells were then loaded onto the gels in cytokine-free media for 20 hours and analyzed via flow cytometry for activation markers and mCherry expression (see FIG. 6C). Activation of cCARiCherry T cells was observed, peaking at lOpM PolySTAT concentration, as evident by both mCherry production and CD69 expression (see FIG. 6D). A lower percentage of mCherry⁺ and CD69⁺ T cells were recorded on FMA- PolySCRM-loaded gels, and the percent of CD69⁺ mock cells remained at baseline, regardless of gel (see FIG. 6D). Although PolySTAT was able to activate cCARiCherry T cells, the levels of activation were low by both metrics. As a result, a series of improvements were made to PolySTAT to enhance CAR binding and cell activation.

[0312] The first step to improve PolySTAT was to enhance CAR binding by synthesizing a new FL monomer with better FL presentation and hydrolytic stability. The commercially available FMA monomer uses a phenyl ester linkage that prevents one of the phenols from being available for CAR binding and is a well-known hydrolysable linker used in drug delivery. See Das et al., Polym. Chem. 7:826-837, 2016. Therefore, FL was conjugated to a methacrylate monomer with a pendant primary amine via an isothiocyanate reaction (see FIG. 7) to form a more stable N,N’-disubstituted urea bond that is commonly used to conjugate FL to proteins. This new FL monomer, 2-propenoic acid, 2-methyl-2- [[[(3’6’-dihydroxy-3-oxospiro[i-sobenzofuran-l(3H),9’-[9H]xanthen]-5- yl)amino]thioxomethyl]amino]ethyl ester or “AEMA-FL” (see FIG. 3A) was confirmed by proton NMR spectroscopy. AEMA-FL-PolySTAT and AEMA-FL-PolySCRM with statistically incorporated 4% FL content were made using the same synthesis steps as FMA- PolySTAT above (see FIG. 8). The % FL/number of FL attached to copolymers were measured by NMR. The fibrin-binding ability of AEMA-FL-PolySTAT was then confirmed in vitro by rotational thromboelastometry (ROTEM). Lamm etal., ACS Biomaterials Sci. Eng. 6:7011-7020, 2020; Chan et al., ACS Biomater. Sci. Eng. 2:403-408; 2016. AEMA-FL- PolySTAT binding to fibrin in vivo was also confirmed by confocal microscopy imaging of blood clots harvested from rats infused with AEMA-FL-PolySTAT or AEMA-FL-PolySCRM.

[0313] An ELISA assay termed PolySTAT ELISA (PS-ELISA) was developed for rapid evaluation of PolySTAT designs. In PS-ELISA, fibrin gels with FMA-PolySTAT or AEMA-FL-PolySTAT at various concentrations were made and exposed to a biotinylated anti- FL antibody. Antibody binding to PolySTAT-bound fibrin gels was then assessed using a streptavi din-conjugated horseradish peroxidase (HRP) in the presence of a fluorogenic peroxidase substrate. Relative fluorescence unit (RFU) outputs indicated peak antibody signal on 5 pM gels, so this PolySTAT concentration was used for subsequent studies. The PS-ELISA was conducted with AEMA-FL PolySTAT, FMA-PolySTAT, and their respective negative controls. Significantly higher antibody binding to AEMA-FL-PolySTAT over FMA- PolySTAT (7’=0.0021) and negative controls was observed see FIG. 3A). Although specific antibody binding to FMA-PolySTAT was observed, the data suggest that the AEMA-FL monomer bound more robustly than the FMA monomer, and we therefore AEMA-FL was used for future studies.

[0314] One of the issues with FMA-PolySTAT and AEMA-FL PolySTAT was limited water solubility due to the inclusion of hydrophobic fluorescein-containing monomers. To counter this, HEMA comonomers in the polymer backbone were replaced with glycerol monomethacrylate (GmMA) (see FIG. 3B). Recently, it was reported that substituting HEMA monomers with GmMA monomers enhanced the water solubility of PolySTAT by incorporating more hydroxyl groups into the polymer. See Lamm et al., ACS Biomaterials Sci. Eng. 6:7011-7020, 2020. It is believed that the more hydrophilic GmMA would both increase the total polymer solubility and also reduce fluorescein - stacking with the tyrosines of FBP, resulting in better presentation of fluorescein for CAR T cell binding. In PS-ELISA, a statistically significant ( ><0.0001) 1.5-fold increase of antibody binding to the GmMA version of AEMA-FL-PolySTAT was observed compared with the HEMA version (see FIG. 3B). Therefore, all future AEMA-FL-Poly STATs were synthesized with a GmMA backbone.

[0315] For further design optimization of PolySTAT, both AEMA-FL content and polymer structure were varied. For AEMA-FL content, it was believed that increasing the number of FL molecules per polymer would increase the number of binding sites and improve avidity of binding to the antigen-binding domain. To this end, a series of AEMA-FL- PolySTATs were synthesized with a range of FL content (2%, 4%, and 10%). Proton NMR and UV-Vis measurements confirmed the FL content in each polymer. However, PS-ELISA results indicated no improvement in antibody binding with the incorporation of more FL monomer in PolySTAT (see FIG. 3C).

[0316] For polymer structure, it was believed that moving from a statistical- incorporation of FL throughout the PolySTAT backbone to a block-copolymer, where the AEMA-FL is concentrated in an “activating block” that is separate from the fibrin-binding domain of PolySTAT, would promote receptor clustering and enhance the CAR T cell response. Wang et al., Cancer Immunol. Immunother. 71 :2801-2814, 2022. To this end, a series of block copolymers were synthesized with discrete segments of AEMA-FL monomers (see FIG. 3D, FIG. 9). For block polymers, macroCTAs or the “activating blocks” with degrees of polymerization (DP) of 13-20 containing varying ratios of GmMA to AEMA-FL (80:20, 60:40, and 70:30) were synthesized via RAFT polymerization. These macroCTAs were then chain extended with GmMA and NHSMA to create block copolymers of similar size and AEMA-FL content (2%, 4%, and 10%) as the previous statistical copolymers. Characterization by gel permeation chromatography (GPC) refractive index (RI) traces indicated successful chain extension of macroCTAs. Finally, the fibrin-binding peptides were conjugated via the NHS-handle. These polymers were tested by PS-ELISA and compared with their respective Poly SCRM negative controls. Interestingly, the addition of more AEMA- FL monomer to the polymer decreased antibody binding (see FIG. 3D). It was observed during the synthesis of the activating block that the AEMA-FL preferentially copolymerizes with itself over reacting with GmMA at higher loading of AEMA-FL, which will result in increased segments of homopolymerized AEMA-FL. Due to the multiple protonation states of fluorescein (see Le Guem et al. , hit. J. Mol. Sci. 21 : 9217, 2020), the local pKa in the immediate area of the homopolymerized blocks of AEMA-FLs could be different from the bulk solution the block copolymers are in, resulting in fluoresceins that have reduced binding to the CAR. In contrast, AEMA-FL are much more spaced out in the statistical copolymers, less able to affect the protonation state of other AEMA-FLs, and therefore retain binding affinity to the CAR

Quantitative Analysis of cCARiCherry T Cells Shows Varied Reactivity to Different AEMA-FL PolySTA T Variants

[0317] A large batch of cCARiCherry CAR T cells with high CAR expression efficiency were generated for in vitro cell activation studies with our panel of AEMA-FL- PolySTAT polymers. For each PolySTAT and PolySCRM, 2%, 4%, and 10% FL in both statistical and block copolymer formats were assessed. cCARiCherry CAR T cells or mock cells were added onto the polymer-incubated gels in cytokine-free media, and images of the cells were taken every 2 hours in an IncuCyte cell imager for 20 hours. Red image mean analyses of the images revealed upregulated mCherry expression within four hours of being added to gels, with peak expression occurring around 8 hours after addition to gels, followed by a plateaued expression level for the remainder of the experiment (see FIG. 4A). As expected, mock cells produced no mCherry signal under any conditions. The images also indicate the highest mCherry expression in cells stimulated with the 2% FL statistical polymer. This observation was corroborated by mCherry and CD69 expression in cells harvested at the end of the 20-hour incubation and analyzed with flow cytometry (see FIG. 4B, 4C). Each block polymer resulted in about 50% mCherry⁺ and 75% CD69⁺ cells, with lower FL content inducing slightly higher CD69 expression. For the statistical polymers, there was a clear inverse relationship between FL content and activation of the cells by both metrics. With about 60% m Cherry cells and CD69 expression over 80%, the 2% statistical AEMA-FL-PolySTAT elicited higher cell activation than the other polymers and comparable activation levels to positive controls. Mock or cCARiCherry T cells were then added onto plain or 2% statistical AEMA-FL-PolySTAT or PolyS CRM-loaded gels for 24 hours and imaged by confocal microscopy. Imaging confirmed mCherry-expressing cells distributed across PolySTAT gels but not plain gels (see FIG. 4D). PolySCRM gels show some cells expressing mCherry, which is likely due to residual PolySCRM left on the gels after their fabrication and PBS washes. Higher magnification (40X) imaging of PolySTAT gels show mCherry-expressing cCARiCherry T cells engaging the PolySTAT matrix.

AEMA-FL-PolySTAT Binds to Fibrin in the ECM of BT-20 Tumors Following Intravenous Injection

[0318] With the evidence that PolySTAT drives robust cCARiCherry activation in vitro, it was next confirmed that the polymer accumulates at tumor fibrin deposits after systemic administration and locally stimulates the cells in tumors. To this end, five million BT-20 cells, a slow-growing human breast cancer cell line that forms tumors with documented fibrin structures in the tumor microenvironment (see Starmans el al., Mol. Pharm. 12: 1921- 1928, 2015), were engrafted subcutaneously in the left flank of NSG mice. After allowing the tumors to grow for 4 weeks, PBS, 2% AEMA-FL-PolySTAT, or the equivalent PolySCRM control were injected intravenously. Tumors were harvested 24 hours later and co-stained with anti-FL-AF488 (to enhance the FL signal from the polymer) and an anti-fibrinogen antibody. Immunofluorescence (IF) images showed extensive FL signal in tumors from mice that received PolySTAT but no FL signal in tumors from mice that received PolySCRM. AF647 (fibrinogen) and AF488 (PolySTAT) signals were analyzed in Image J (Fiji) using the JACoP plugin to quantify signal overlap from these two layers. Distance-based colocalization analyses from six images between two tumors provided an average PolySTAT on fibrinogen overlap of 92.7% with a standard deviation of 4.8%. The same tumors were also co-stained with anti-FL AF488 and anti-CD31 AF647 antibodies. Confocal images indicated that PolySTAT depositions were also found near vascularized sections of the tumors. Collectively, these data indicate that PolySTAT will accumulate in well vascularized tumors with fibrin deposits. cCARiCherry T Cells Are Activated and Express mCherryffluc Locally in BT-20 Tumors Following Administration of AEMA-FL-Poly ST AT

[0319] Finally, whether cCARiCherry T cells can interact with tumor bound PolySTAT and turn on iSynPro was evaluated. To do this, five million BT-20 cells were engrafted subcutaneously on the left flank of NSG mice. After 4 weeks, 10⁶ cCARiCherry or mock T cells were injected IV into the mice (n=4 per group). Two days (48 hours) later, PBS or lOmg/kg of 2% AEMA-FL-Poly STAT or PolySCRM were injected IV. Mice were imaged with a Xenogen IVIS imaging machine to monitor induction of iSynPro-regulated mCherry:ffluc production (see FIG. 5A). IVIS images over this period indicated transient ffluc expression primarily limited to the engrafted flank tumor, peaking within the first 24 hours, in mice that received PolySTAT and cCARiCherry T cells. Minimal signal was observed in mice that received cCARiCherry T cells and PolySCRM or PBS. Outside of the flank tumor, activated cells appear to have accumulated in the heads of most PolySTAT-treated mice. This is likely an indicator of brain metastases, a very common metastasis destination for BT-20 cells. See Jin et al., Nature 588:331-336, 2020. At each time point with significance by oneway ANOVA, (p< 0.05), Tukey’s HSD post-hoc analysis was performed between each group. Statistical significance was found between the cCARiCherry + PolySTAT group and the cCARiCherry + PBS at t=24 hours (p<0.0001), t=48 hours (p<0.0001), t=72 hours (/?<0.0001), t=96 hours (p=0.0003), t=120 hours (p=0.0233), and t=144 hours (/?=0.0150). Statistical significance was also found between the cCARiCherry + PolySTAT and mock + PolySTAT groups at t=24 hours (p<0.0001), t=48 hours (p 0.0001 ), t=72 hours (p 0.0001), t=96 hours (p=0.0034), t=l 20 hours ( 2=0.0106), and t=144 hours (/?=0.0113), and t=168 hours ( =0.0219). These results demonstrate for the first time spatially restricted CAR T cell activation controlled by a polymer that accumulates in the tumor microenvironment.

[0320] There was only significant difference between cCARiCherry|PolySCRM and cCARiCherry |PBS groups (p=0.0201 ) at the 24-hour time point, despite PolySCRM, identical to PolySTAT but with a scrambled FBP sequence, being injected after the T cells. Thus, circulating FL-conjugated polymers have limited impact on circulating T cell activation, reducing toxicity concerns associated with possible PolySTAT re-doses. This also suggests that the order of T cell or polymer injection does not matter. The later claim was supported with a second in vivo experiment switching the order of injections so that polymer is administered to the animal before T cells. In this experiment, five million BT-20 cells were engrafted subcutaneously on the left flank of NSG mice. After 4 weeks, lOmg/kg of 2% AEMA-FL-PolySTAT or PolySCRM were injected IV. One day (24 hours) later, 10⁶ cCARiCherry or mock T cells were injected IV into the mice (n=3 per group). Mice were imaged with a Xenogen IVIS imaging machine to monitor induction of iSynPro-regulated mCherry:ffluc production 8 hours, 24 hours, and every subsequent 24 hours after T cell injections. All mice had high non-specific signal for the first 24-48 hours following T cell injections with concentrated flux in the lungs. This transient non-specific ffluc expression is characteristic of iSynPro function following cell injections and concentrating in the lungs is characteristic of CAR T cell migration following IV injections. See Skovgard etal., Mol. Ther. Oncolytics 22:355-367, 2021. Flux from the tumors was quantified and unpaired one-way T tests with Welch’s correction for unequal variance was conducted at each time point (see FIG. 10). Statistical significance was found between the cCARiCherry + Poly STAT and the cCARiCherry + PolySCRM groups at t=24 hours (p=0.0255), t=48 hours (p=0.0428), t=72 hours (p=0.0257), t=96 hours (p=0.0473), t=120 hours (p=0.0127), and t=144 hours (p=0.0389).

Discussion

[0321] IF-THEN gated systems can be used to guide the effector function of CAR T cell therapies by spatially differentiating tumors from healthy tissues. In vivo spatial control of CAR T cell response can alleviate CAR specificity issues, such as on-target, off-tumor toxicity. Because tumor-specific antigens are rare, CARs usually target tumor-associated antigens (TAAs). Although these antigens are highly expressed on the cancer cells, they may also be found on select healthy tissues, potentially rendering those tissues susceptible to CAR T cell-inflicted damage. While CAR T cell-related toxicities are currently managed by antiinflammatory drugs such as corticosteroids or tocilizumab (see Gardner et al., Blood 128:586- 586, 2016), therapeutic CAR T cell strategies with more specificity are preferred. To this end, the present study’s IMPACT system — utilizing a dual promoter construct with a constitutively expressed anti-FL CAR and inducible mCherry:ffluc fusion protein (cCARiCherry) — was created. The stringent, switch-like function of cCARiCherry T cells allows for conditional expression of mCherry:ffluc (“THEN” outcome) when provided a spatially-restricted “IF” condition. For this, fluorescein-modified PolySTAT was utilized to bind fibrin deposits in irregular tumor vasculature and provide an epitope for fluorescein-specific CARs. In this work, the presentation and linker chemistry of FL tags on PolySTAT were altered to maximize CAR binding and cCARiCherry T cell activation in vitro and in vivo.

[0322] Anti-fluorescein, or anti-FITC, CARs are a type of universal CAR with specificity for fluorescein-modified intermediate adapter molecules. Being one of the first universal CARs developed, a multitude of different adaptor molecules have effectively been used with anti-FL CARs, resulting in similar or improved performance when compared with benchmark CAR T cells. See Kim et al., J. Am. Chem. Soc. 137:2832-2835, 2015; Ma et al., Proc. Natl. Acad. Sci. U.S.A. 113:E450-E458, 2016; Tamadac/ ci/., Clin. Cancer Res. 18:6436- 6445, 2012; Cao et al., ACS Synth. Biol. 10: 1176-1183, 2021. This success has led to the opening of ENGLIGHTen-01, the first in-human clinical trial with a universal CAR therapy, which pairs an anti-FITC CAR with FITC-folate to target folate receptor⁺ (FOLR1⁺) osteosarcoma. See Albert et al., J. Clin. Oncol. 41:TPS11581-TPS11581, 2023. The development of multiple targeting molecules has also translated into multiplexed targeting abilities with the anti-FL CAR, leading to enhanced reactivity against heterogenous tumors. See Lee et al., Cancer Res. 79:387-396, 2019. Recently, a pair of FL-conjugated intermediate adaptor molecules were developed that integrate into the cell membranes of target cells, opening up the opportunity for even broader tumor targeting capabilities using a single cell product. See Jensen & James, “Phospholipid ether (PLE) CAR T cell tumor targeting (CTCT) agents,” WorldPatent, 2018; Zhang et al., Nat. Biomed. Eng., 2023, doi: 10.1038/s41551-023- 01048-8. Although adaptor molecules can be injected locally or have shown preferential retention in malignant cells (see Weichert et al., Sci. Transl. Med. 6:240ra75, 2014), this generalized targeting method creates a high risk for on-target, off-tumor toxicity. As CAR T cells are used for more expansive cancer targeting applications, measures need to be taken for CAR T cells to spatially differentiate malignant and healthy tissues.

[0323] IF-THEN gating can be used to add a layer of specificity to differentiate malignant from healthy tissues. With CAR T cell therapies, this gate can be created with an inducible promoter that drives a therapeutic transgene when provided an “IF” condition. One inducible promoter platform, SynNotch, has been used to regulate the expression of different therapeutic transgenes, including a CAR (see Moghimi et al., Nat. Commun. 12:511, 2021; Srivastava et al., Cancer Cell 35:489-503. e8, 2019; Choe et al., Sci. Transl. Med. 13, 2021), but it requires orthogonal promoters and large sensor-transcription factor proteins, thereby rendering viral encoding cumbersome. Other inducible promoters consisting of repeated endogenous transcription response elements (TREs), such as repeated NF AT TREs, have been used to drive exogenous cytokine secretion following CAR or TCR stimulation. See Hooijberg et al., Blood 96:459-466, 2000; Zhang et al., Mol. Ther. 19:751-759; 2011. However, such promoters are limited in their reactivity due to their reliance on a single transcription factor. To address this limitation, others have made more complex synthetic promoters by adding multiple different TRE’s upstream from a minimal (core) promoter. See Blazeck & Alper, Biotechnol. J. 8:46-58, 2013. One of these more complex systems, inducible synthetic promoter (iSynPro), was used in the present study’s IF-THEN gating system due to its low genetic profile, stringent off-state, and strong induction following stimulation with antigen.

[0324] With the IMPACT platform, a spatial distinction is made between cancerous and healthy tissues by labeling irregular tumor ECM rather than a cell marker to provide the “IF” condition for iSynPro induction. The present study targeted a tumor characteristic rather than an antigen directly on cancerous cells to enable in situ cell activation with no possibility that the anti-FL CAR (cCAR) could engage tumors directly. Targeting abnormal tumor ECM characteristics also provides a degree of universality unavailable if a specific TAA is targeted, as this approach can be broadly applied across different tumors. Furthermore, PolySTAT needed to bind to a tumor attribute that is relatively static temporally and spatially to ensure adequate retention time in tumors. Therefore, targeting structural components of the ECM were preferential to soluble targets or cell receptors, which are subject to degradation, metabolization, and internalization. In this work, PolySTAT was used to target fibrin, as it is a well-documented component of solid tumor ECMs. See Dvorak, N. Engl. J. Med. 315: 1650- 1659, 1986. Fibrin matrices are able to form networks in the ECM of many tumors due to the high permeability of tumor microvasculature (see Dvorak et al., Cancer Res. 44:3348-3354, 1984), which allows substantial fibrinogen to extravasate into the ECM where it is rapidly clotted (see Dvorak, supra . As tumors expand, new fibrin matrices form at sites of growth and older fibrin matrices transform into vascularized and collagenous matrices, characteristic of mature tumor stroma. See Folkman, Adv. Cancer Res. 43: 175-203, 1985. This paradigm results in relatively constant fibrin distribution patterns throughout tumors, thereby making fibrin a viable marker of tumor ECM. This also indicates the possible utility of tailoring PolySTAT to target other solid tumor attributes, such as collagen. Like fibrin, collagen is abundant in a variety of tumors, having a role in their growth and ability to metastasize. See Nerenberg et al. , Cancer Genomics Proteomics 4 :319-328, 2007; Winkler etal., Nat. Commun. 11 :5120, 2020. As a result, other groups have used collagen as a target for various cancer therapies. See Liang et al., Sci. Rep. 6: 18205, 2016; Baldari et al., Cancers (Basel) 14:4706, 2022. By replacing our fibrin-binding peptide with one of the many collagen-binding peptides reported, PolySTAT can be re-directed as desired. See Anderson et al., Nano Lett. 22:4182- 4191, 2022; Ezeani etal., J. Am. Heart Assoc. 10:e022139, 2021. Similarly, the extra-domain B (EDB) splice variant of fibronectin is strongly associated with solid malignancies in adults (see Rick etal., Semin. Oncol. 46:284-290, 2019) and has recently been used as a cancer drugdelivery target (see Kumra & Reinhardt, Adv. Drug Del iv. Rev. 97: 101-110, 2016). Further demonstrating the modularity of PolySTAT, EBD-fibronectin could be used as another target for PolySTAT in the IMPACT system.

[0325] There were several characteristics that were considered when designing PolySTAT for the IMPACT system. In addition to modifications made to improve FL presentation and hydrolytic stability, polymer structure and molecular weight were also considered, as they are main factors determining the circulation half-life of polymers. The polymers tested in this work were 43.8-47.5 kDa, which is a similar size to PolySTAT used in our hemostasis work. Biodistribution studies in rats with radiolabeled PolySTAT revealed initial distribution half-life of 20 min and elimination half-life of 14.4 hours, with >50% of PolySTAT cleared from the body within 1 hour through renal clearance. See Chan et al., Sci. Transl. Med. 7:277ra29-277ra29, 2015. PolySTAT did not accumulate in the heart and lungs at any time. See id. While soluble PolySTAT is rapidly eliminated, PolySTAT extravasated and penetrated the tumor effectively, binding to fibrin throughout the tumor microenvironment. The PolySTAT size used in these studies is well-suited for CAR T cell activation; smaller polymers would reduce fibrin-binding valency and shorten the therapy’s half-life, but larger polymers would reduce the polymer’s ability to extravasate and diffuse through the tumor.

[0326] The CAR epitope presented on PolySTAT can also be modulated to accommodate other universal CARs and further enhance tumor specificity. Namely, PolySTAT can be modified to replace the current CAR epitope, FL, with the cognate target of one of the many other reported universal CARs. See Urbanska et al., Cancer Res. 72:1844- 1852, 2012; Rodgers et al., Proc. Natl. Acad. Set. USA 113:E459-E468, 2016; Minutolo etal., J. Am. Chem. Soc. 142:6554-6568, 2020. The CAR epitope can also be modified to have a “mask” that is removed upon exposure to the TME or an external stimulus. Tumor microenvironments are known to have upregulated MMP activity, reactive oxygen species, and acidic conditions, all of which can be taken advantage of when designing an antigen mask. See Yin et al., Methods Enzymol. 657:59-87, 2021. In the case of fluorescein, one could leverage self-immolative linkers such as difluorophenyl esters or p-aminobenzlyalcohol attached to an MMP-cleavable peptide, ROS-cleavable boronate fluorescent probe, or a caged fluorescein that requires UV light to unmask the phenols in the binding domain. Huang et al., Anal. Chem. 92: 15463-15471, 2020; Richard et al., Bioconjug. Chem. 19: 1707-1718, 2008; Miller et al., J. Am. Chem. Soc. 127: 16652-16659, 2005; Kobayashi etal., J. Am. Chem. Soc. 129:6696-6697, 2007. Similar modifications could feasibly be made for other universal CAR targets

[0327] Additional safety interventions can be used to complement the IMPACT system. Once activated, iSynPro does not return to an off state for several days, and this timeline is extended if the cells continue to be stimulated. Furthermore, if the iSynPro- restricted transgene is another CAR, stimulation via that CAR can induce a feedback loop that self-perpetuates its own expression. If this phenomenon occurs outside the TME, toxicities can ensue. Fortunately, this loop can be broken with the inclusion of a suicide gene on the CARs, which degrade the receptor upon exogenous delivery of a suicide switch-inducing drug. A more interesting approach may be the use of dasatinib, a tyrosine kinase inhibitor, which has previously been used turn off CAR T cell function. See Mestermann et al., Set. Transl. Med. l l :eaau5907, 2019. This can be applied to the system of this study, as dasatinib blocks the intracellular signaling pathways responsible for iSynPro induction. Dasatinib can therefore be used as a manual switch to block the “IF” condition if appropriate.

[0328] While the functionality of IMPACT was demonstrated here, there are a few limitations to the system. First, IMPACT in the current iteration necessitates the presence of fibrin deposits near tumors, thereby excluding all liquid cancers, such as leukemias and lymphomas, from consideration for this therapeutic strategy. Another possible limitation to this iteration of IMPACT is that there are scenarios where tumors are not the only sites of fibrin deposits. Because CAR T cells will become activated in regions of high tissue regeneration due to PolySTAT, IMPACT may not limit cell activation to tumors in patients who recently underwent a major surgery or have a condition associated with upregulated fibrin deposition, such as progressed arthritis. See Hiigle et al., EBioMedicine 81 : 104081 , 2022. Although fibrin deposits will dissolve as wounds heal, it may not be in the patient’s best interest to delay the commencement of treatment if the tumor is aggressive.

[0329] Collectively, this work builds the foundation for the IMPACT system to enhance the safety and specificity of universal CAR therapies. This strategy uses novel conditional requirements to anatomically control expression of a therapeutic payload. The IF- THEN gate imposed with IMPACT first employs a synthetic biomaterial to mark a specific anatomic region (tumors) for the CAR T cells to activate. The CAR T cells are then primed and able to express a multitude of tumor targeting agents, such as a BiTE (see Staerz et al., supra) or another CAR. Due to the synthetic and modular nature of IMPACT, various pieces of the system can be refined to accommodate the situational requirements of different patients. Although these data are proof-of-concept, this work demonstrates the framework of a robust system with a plethora of new avenues for future engineered cell (e.g. , CAR T cell) approaches. Moving forward, a therapeutic payload should be placed under iSynPro control and toxicity/therapeutic outcome should be compared with an equivalent treatment without the IMPACT system. Testing different payloads, and therefore different types of therapies, will demonstrate the versatility of this system. Example 2: cCARiCherry T Cells Can Be Serially Stimulated, and Cell Function Can Be Modulated With Dasatinib

[0330] The potential to sequentially activate the CAR T cells in the IMPACT system to model PolySTAT redosing was explored. To do this, cCARiCherry T cells were stimulated and characterized on PolySTAT gels as performed above. The activated cells were then dosed with dasatinib, an FDA-approved small molecule tyrosine kinase inhibitor, to reversibly block CAR T cell functions by blocking CD3(^ signaling through LCK phosphorylation inhibition. After 48 hours the cells were re-analyzed, and dramatically reduced CD69 and mCherry expression were noted, indicating that the cells were rendered to an inactive state. To explore whether the IMPACT system can respond to a second stimulus after deactivation, dasatinib- treated cells were re-stimulated on AEMA-FL-PolySTAT-loaded gels. Re-stimulated cells showed a significant re-expression of mCherry and CD69 to levels comparable with that observed during the first stimulation, suggesting that iSynPro can be robustly re-induced to express its payload in response to a second stimulus. Collectively, these results both show that cCARiCherry T cells can be serially stimulated and that dasatinib has the potential use as an extra safety measure to control cCARiCherry T cell function.

Example 3; cCARiCherry T cells Are Responsive to PolySTAT in Different Fibrin Clot Models

[0331] Transitioning to an in vivo context, PolySTAT can engage with fibrin in one of two ways: integrating into actively forming fibrin clots (as has been modeled so far with “mixed gels”), or “coating” pre-existing fibrin clots in the body. To ensure that both scenarios would drive similar activation in cCARiCherry T cells, fibrin gels were pre-formed in vitro and the gels were coated with PolySTAT for one hour. Gels were then washed and cCARiCherry T cells were added onto these “coated” gels or standard “mixed” gels. Results showed no difference in either CD69 expression or mCherry production between these two models, indicating that this system will function similarly in both biological contexts.

[0332] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for treating a solid tumor cancer, the method comprising: administering to a subject having the solid tumor cancer an effective regimen of

(i) a universal chimeric antigen receptor (CAR) epitope, and

(b) a CAR immune cell comprising

2. The method of claim 1, wherein the universal CAR epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope.

3. The method of claim 1, wherein the universal CAR epitope is a masked epitope comprising a mask that is removed upon exposure to the tumor microenvironment.

4. The method of claim 3, wherein the mask comprises a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide.

5. The method of claim 4, wherein the self-immolative linker comprises a difluorophenyl ester or a p-aminobenzylalchohol.

6. The method of claim 3, wherein the universal CAR epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

7. The method of any one of claims 1 to 6, wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrinfibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

8. The method of any one of claims 1 to 6, wherein the polypeptide that specifically binds to the ECM component is a peptide or a single chain antibody.

9. The method of any one of claims 1 to 6, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide.

10. The method of claim 9, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of

X{Ar}XCPY{G/D}LC{Ar}IX (SEQ ID NO:1), wherein X = any amino acid, Ar = aromatic amino acid, and D/G = Gly or Asp;

XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid; and NHGCYNSYGVPYCDYS (SEQ ID NO:3), wherein X = any amino acid.

1 1 . The method of claim 9, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of Ac-Y{DGl}C{HPr} (Y(3-C1)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac=N- terminus acetylation, DGl=D-glutamic acid, HPr=hydroxyproline, Y(3-C1) = 3- chlorotyrosine, and NH2 = C terminus amidation; and

Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID NO:5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation.

12. The method of any one of claims 1 to 6, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a collagen- binding peptide.

13. The method of claim 12, wherein the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of

(GP{HPr})7-(D)e (SEQ ID NO:6), wherein HPr = hydroxyproline;

(G{P(4-Fl)}{Hpr})_n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9;

(GPP)s (SEQ ID NO: 8);

(P{HPr}G)7 (SEQ ID NO:9), wherein HPr = hydroxyproline;

TLTYTWS (SEQ ID NO: 10);

WYRGRL (SEQ ID NO: 11);

TKKLTLRT (SEQ ID NO: 12);

TKKTLRT (SEQ ID NO: 13);

LRELTLNNN (SEQ ID NO: 14); and

LRELHLNNN (SEQ ID NO: 15).

14. The method of any one of claims 1 to 6, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4-1BB signaling domain and a CD28 signaling domain.

15. The method of any one of claims 1 to 6, wherein the encoded anticancer therapeutic protein is selected from the group consisting of

(i) a second CAR that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells,

(ii) an antibody that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells,

(iii) an immune checkpoint inhibitor, and

(iv) an immunostimulatory cytokine.

16. The method of claim 15, wherein the encoded therapeutic protein is the second CAR that specifically recognizes a tumor-associated antigen expressed by the solid tumor cells.

17. The method of claim 15, wherein the encoded therapeutic protein is the antibody the specifically recognizes a tumor-associated antigen expressed by the solid tumor cells.

18. The method of claim 17, wherein the antibody is a bispecific antibody.

19. The method of claim 17, wherein the antibody is a single-chain antibody.

20. The method of claim 18, wherein the bispecific antibody is a bispecific T cell engager.

21. The method of claim 15, wherein the encoded therapeutic protein is the immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4, anti-PD-l/PD-Ll, and a combination thereof.

22. The method of claim 15, wherein the encoded therapeutic protein is the immunostimulatory cytokine, optionally wherein the immunostimulatory cytokine is selected from the group consisting of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-alpha (IFN-a), interleukin- 12 (IL-12), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

23. The method of any one of claims 1 to 6, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

24. The method of any one of claims 1 to 6, wherein the polymer is administered before the CAR immune cell.

25. The method of any one of claims 1 to 6, wherein the CAR immune cell is administered before the polymer.

26. The method of any one of claims 1 to 6, wherein the solid tumor cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular carcinoma, melanoma, mesothelioma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, basal cell carcinoma, cutaneous squamous cell carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, cervical cancer, endometrial carcinoma, esophageal carcinoma, gastric carcinoma, and Merkel cell carcinoma.

27. The method of any one of claims 1 to 6, wherein the CAR immune cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

28. The method of any one of claims 1 to 6, wherein the polymer is a synthetic polymer.

29. The method of claim 28, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units, optionally wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

30. The method of claim 29, wherein the polymer is a block copolymer or a statistical copolymer.

31. A system for polymer-mediated activation of a CAR immune cell, the system comprising:

(i) a universal chimeric antigen receptor (CAR) epitope, and

(b) a CAR immune cell comprising

32. The system of claim 31, wherein the universal CAR epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope.

33. The system of claim 31, wherein the universal CAR epitope is a masked epitope comprising a mask that is removed upon exposure to the tumor microenvironment.

34. The system of claim 33, wherein the mask comprises a self-immolative linker and/or a matrix metalloproteinase (MMP)-cleavable peptide.

35. The system of claim 34, wherein the self-immolative linker comprises a difluorophenyl ester or a p-aminobenzylalchohol.

36. The system of claim 33, wherein the universal CAR epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

37. The system of any one of claims 31 to 36, wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrinfibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

38. The system of any one of claims 31 to 36, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide.

39. The system of any one of claims 31 to 36, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a polypeptide.

40. The system of any one of claim 39, wherein the polypeptide that specifically binds to the ECM component is a peptide or a single chain antibody.

41. The system of claim 39, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide.

42. The system of claim 41, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of

43. The system of claim 41, wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of

Ac-Y{DGl}C{HPr}{Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac=N- terminus acetylation, DGl=D-glutamic acid, HPr=hydroxyproline, Y(3-C1) = 3- chlorotyrosine, and NH2 = C terminus amidation; and

44. The system of claim 39, wherein the polypeptide that specifically binds to the ECM component of the tumor microenvironment is a collagen-binding peptide.

45. The system of claim 44, wherein the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of

(GP{HPr})7-(D)6 (SEQ ID NO:6), wherein HPr = hydroxyproline;

(G{P(4-Fl)}{Hpr})n (SEQ ID NO:7), wherein P(4-F1) = 4-fluoroproline, HPr = hydroxyproline, and n is an integer from 7 to 9;

(GPP)s (SEQ ID NO:8);

(P{HPr}G)? (SEQ ID NO:9), wherein HPr = hydroxyproline;

TLTYTWS (SEQ ID NO: 10);

WYRGRL (SEQ ID NO: 11);

TKKLTLRT (SEQ ID NO: 12); TKKTLRT (SEQ ID NO: 13);

LRELTLNNN (SEQ ID NO: 14); and LRELHLNNN (SEQ ID NO: 15).

46. The system of any one of claims 31 to 36, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4- IBB signaling domain and a CD28 signaling domain.

47. The system of any one of claims 31 to 36, wherein the encoded therapeutic protein is a second CAR.

48. The system of claim 47, wherein the second CAR specifically recognizes a tumor-associated antigen.

49. The system of claim 47, wherein the second CAR specifically recognizes a second universal CAR epitope.

50. The system of any one of claims 31 to 36, wherein the encoded therapeutic protein is an antibody.

51. The system of claim 50, wherein the antibody is a bispecific antibody.

52. The system of claim 50, wherein the antibody is a single-chain antibody.

53. The system of claim 51, wherein the bispecific antibody is a bispecific T cell engager.

54. The system of claim 50, wherein the antibody is an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor is selected from the group consisting of anti-CTLA-4, anti-PD-l/PD-Ll, and a combination thereof.

55. The system of any one of claims 31 to 36, wherein the encoded therapeutic protein is an immunostimulatory cytokine, optionally wherein the immunostimulatory cytokine is selected from the group consisting of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-alpha (IFN-a), interleukin- 12 (IL-12), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

56. The system of any one of claims 31 to 36, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21 .

57. The system of any one of claims 31 to 36, wherein the CAR immune cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

58. The system of any one of claims 31 to 36, wherein the polymer is a synthetic polymer.

59. The system of claim 58, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units, optionally wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

60. The system of claim 59, wherein the polymer is a block copolymer or a statistical copolymer.

61. A method for expressing a therapeutic protein at the site of a tissue or cell of interest in a subject, the method comprising: administering to a subject an effective regimen of

(i) a universal epitope, and

(b) an engineered cell comprising

(B) an intracellular signaling domain derived from a cell surface receptor, and

62. The method of claim 61, wherein the universal epitope is selected from the group consisting of fluorescein, biotin, di nitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope.

63. The method of claim 61, wherein the universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tissue or cell of interest.

64. The method of claim 63, wherein the mask comprises a self-immolative linker, optionally wherein the self-immolative linker comprises a difluorophenyl ester or a p- aminobenzylalchohol, or optionally wherein the universal epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

65. The method of any one of claims 61 to 64, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide.

66. The method of any one of claims 61 to 64, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a polypeptide, optionally wherein the polypeptide is a peptide or a single chain antibody.

67. The method of any one of claims 61 to 64, wherein the extracellular binding domain is a single chain Fv (scFv).

68. The method of any one of claims 61 to 64, wherein the engineered receptor is a chimeric antigen receptor (CAR).

69. The method of claim 68, wherein the CAR comprises a CD3^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4- IBB signaling domain and a CD28 signaling domain.

70. The method of claim 68, wherein the engineered cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

71. The method of any one of claims 61 to 64, wherein the intracellular signaling domain is derived from an enzyme-linked cellular receptor, optionally wherein the enzyme-linked cellular receptor is selected from the group consisting of an antigen receptor, a cytokine receptor, and a growth factor receptor.

72. The method of claim 71, wherein the enzyme-linked cellular receptor is the antigen receptor, optionally wherein the antigen receptor is a T cell receptor.

73. The method of any one of claims 61 to 64, wherein the engineered receptor is a synNotch receptor in which the intracellular signaling domain comprises a Notch transcriptional regulator that is released from the cell membrane upon engagement of the extracellular binding domain with the universal epitope.

74. The method of any one of claims 61 to 64, wherein the encoded therapeutic protein is selected from the group consisting of an antibody, a second cell surface receptor, a soluble receptor, a cytokine, a chemokine, and a growth factor.

75. The method of claim 74, wherein the antibody is a bispecific antibody, a single-chain antibody, and/or an immune checkpoint inhibitor, optionally wherein the bispecific antibody is a bispecific T cell engager.

76. The method of claim 74, wherein the second cell surface receptor is a second engineered receptor.

77. The system of claim 74, wherein the encoded therapeutic protein is a cytokine, optionally wherein the cytokine is selected from the group consisting of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-alpha (IFN-a), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), and interleukin-21 (IL-21).

78. The method of any one of claims 61 to 64, wherein the therapeutic protein is an immunosuppressive therapeutic protein, optionally wherein the immunosuppressive therapeutic protein is selected from the group consisting of CTLA-4-Fc, TNFR-Fc, and an anti-TNFa antibody.

79. The method of any one of claims 61 to 64, wherein the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment, optionally wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

80. The method of claim 79, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide, optionally wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of

XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid;

NHGCYNSYGVPYCDYS (SEQ ID NO:3), wherein X = any amino acid;

Ac-Y{DGl}C{HPr}{Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac=N- terminus acetylation, DGl=D-glutamic acid, HPr=hydroxyproline, Y(3-C1) = 3- chlorotyrosine, and NH2 = C terminus amidation; and Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID N0:5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation.

81. The method of claim 79, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a collagen-binding peptide, optionally wherein the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of

(GP{HPr})7-(D)e (SEQ ID NO:6), wherein HPr = hydroxyproline;

(GPP)s (SEQ ID NO: 8);

(P{HPr}G)7 (SEQ ID NO:9), wherein HPr = hydroxyproline;

TLTYTWS (SEQ ID NO: 10);

WYRGRL (SEQ ID NO: 11);

TKKLTLRT (SEQ ID NO: 12);

TKKTLRT (SEQ ID NO: 13);

LRELTLNNN (SEQ ID NO: 14); and

LRELHLNNN (SEQ ID NO: 15).

82. The method of claim 68, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

83. The method of any one of claims 61 to 64, wherein the polymer is a synthetic polymer.

84. The method of claim 83, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units, optionally wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

85. The method of claim 84, wherein the polymer is a block copolymer or a statistical copolymer.

86. A system for polymer-mediated activation of an engineered cell, the system comprising:

(i) a universal epitope, and

(b) an engineered cell comprising

(B) an intracellular signaling domain derived from a cell surface receptor, and

87. The system of claim 86, wherein the universal epitope is selected from the group consisting of fluorescein, biotin, dinitrophenyl, 1,3 diketone, Lucifer yellow, dansyl, an AlexaFluor dye, a rhodamine derivative, and a peptide neo-epitope.

88. The system of claim 86, wherein the universal epitope is a masked epitope comprising a mask that is removed upon exposure to the tissue or cell of interest.

89. The system of claim 88, wherein the mask comprises a self-immolative linker, optionally wherein the self-immolative linker comprises a difluorophenyl ester or a p- aminobenzylalchohol, or optionally wherein the universal epitope is fluorescein and the masked epitope comprises an ROS-cleavable boronate fluorescent probe or a caged fluorescein that requires UV light to unmask the phenols in the binding domain.

90. The system of any one of claims 86 to 89, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a vitamin, an oligonucleotide, a sugar, a lipid, or a polypeptide.

91. The system of any one of claims 86 to 89, wherein the moiety that specifically binds to the component of the tissue or cell of interest is a polypeptide, optionally wherein the polypeptide is a peptide or a single chain antibody.

92. The system of any one of claims 86 to 89, wherein the extracellular binding domain is a single chain Fv (scFv).

93. The system of any one of claims 86 to 89, wherein the engineered receptor is a chimeric antigen receptor (CAR).

94. The system of claim 93, wherein the CAR comprises a CD3(^ signaling domain and a costimulatory signaling domain selected from the group consisting of a 4- IBB signaling domain and a CD28 signaling domain.

95. The system of claim 93, wherein the engineered cell is a CAR T cell, a CAR macrophage, a CAR neutrophil, or a CAR NK cell.

96. The system of any one of claims 86 to 89, wherein the intracellular signaling domain is derived from an enzyme-linked cellular receptor, optionally wherein the enzyme-linked cellular receptor is selected from the group consisting of an antigen receptor, a cytokine receptor, and a growth factor receptor.

97. The system of claim 96, wherein the enzyme-linked cellular receptor is the antigen receptor, optionally wherein the antigen receptor is a T cell receptor.

98. The system of any one of claims 86 to 89, wherein the engineered receptor is a synNotch receptor in which the intracellular signaling domain comprises a Notch transcriptional regulator that is released from the cell membrane upon engagement of the extracellular binding domain with the universal epitope.

99. The system of any one of claims 86 to 89, wherein the encoded therapeutic protein is selected from the group consisting of an antibody, a second cell surface receptor, a soluble receptor, a cytokine, a chemokine, and a growth factor.

100. The system of claim 99, wherein the antibody is a bispecific antibody, a single-chain antibody, and/or an immune checkpoint inhibitor, optionally wherein the bispecific antibody is a bispecific T cell engager.

101. The system of claim 99, wherein the second cell surface receptor is a second engineered receptor.

102. The system of claim 99, wherein the encoded therapeutic protein is a cytokine, optionally wherein the cytokine is selected from the group consisting of interleukin-2 (IL-2), interferon-alpha (IFN-a), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), and interleukin-21 (IL-21).

103. The system of any one of claims 86 to 89, wherein the therapeutic protein is an immunosuppressive therapeutic protein, optionally wherein the immunosuppressive therapeutic protein is selected from the group consisting of CTLA-4-Fc, TNFR-Fc, and an anti-TNFa antibody.

104. The system of any one of claims 86 to 89, wherein the component of the tissue or cell of interest is an extracellular matrix (ECM) component of a tumor microenvironment, optionally wherein the ECM component of the tumor microenvironment is selected from the group consisting of fibrin, collagen, a fibrin-fibronectin complex, the extra-domain B (EBD) splice variant of fibronectin, an N-terminal fibronectin type I domain, tenascin-C, heparan sulfate, and heparin.

105. The system of claim 104, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a fibrin-binding peptide, optionally wherein the fibrin-binding peptide comprises an amino acid sequence selected from the group consisting of

XXCXYYGTCLX (SEQ ID NO:2), wherein X = any amino acid;

NHGCYNSYGVPYCDYS (SEQ ID NO:3), wherein X = any amino acid;

Ac-Y{DGl}C{HPr}{Y(3-Cl)}GLCYIQGK-NH2 (SEQ ID NO:4), wherein Ac = N- terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, Y(3-C1) = 3- chlorotyrosine, and NH2 = C terminus amidation; and Ac-W{DGl}C{HPr}WGLCWIQGK-NH2 (SEQ ID N0:5), wherein Ac = N-terminus acetylation, DG1 = D-glutamic acid, HPr = hydroxyproline, and NH2 = C terminus amidation.

106. The system of claim 104, wherein the moiety that specifically binds to the ECM component of the tumor microenvironment is a collagen-binding peptide, optionally wherein the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of

(GP{HPr})7-(D)e (SEQ ID NO:6), wherein HPr = hydroxyproline;

(GPP)s (SEQ ID NO: 8);

(P{HPr}G)7 (SEQ ID NO:9), wherein HPr = hydroxyproline;

TLTYTWS (SEQ ID NO: 10);

WYRGRL (SEQ ID NO: 11);

TKKLTLRT (SEQ ID NO: 12);

TKKTLRT (SEQ ID NO: 13);

LRELTLNNN (SEQ ID NO: 14); and

LRELHLNNN (SEQ ID NO: 15).

107. The system of claim 93, wherein the inducible promoter has the nucleotide sequence shown in SEQ ID NO:21.

108. The system of any one of claims 86 to 89, wherein the polymer is a synthetic polymer.

109. The system of claim 108, wherein the plurality of repeating units forming the polymer backbone comprises hydrophilic repeating units, optionally wherein the hydrophilic repeating units are selected from the group consisting of carboxybetaines, sulfobetaines, phosphobetaines, (hydroxy-ethyl)methacrylate (HEMA), glycerol monomethacrylate, (GmMA), and N-(2-hydroxypropyl)methacrylamide (HPMA).

110. The system of claim 109, wherein the polymer is a block copolymer or a statistical copolymer.