Chimeric antigen receptor (CAR) modulation

1. A polypeptide comprising, from N-terminus to C-terminus:

a) an extracellular binding domain comprising an antigen-binding fragment of an antibody;

b) a transmembrane domain;

c) a first intracellular signaling domain comprising an intracellular domain of a costimulatory molecule;

d) a repressible protease; and

e) the combination of A) an immunoreceptor tyrosine-based activation motif (ITAM) containing primary cytoplasmic signaling sequence, and B) a second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule; wherein the ITAM containing primary cytoplasmic signaling sequence and the second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule can be in any N-terminus to C-terminus order relative to each other.

2. The polypeptide of claim 1 , further comprising a cofactor for the repressible protease and at least one protease cleavage site of the repressible protease.

3. The polypeptide of claim 2 , wherein:

a) the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3);

b) the cofactor is an HSV NS4A domain; and/or

c) the at least one protease cleavage site is located:

i) between the transmembrane domain and the first intracellular signaling domain comprising an intracellular domain of a costimulatory molecule;

ii) between the first intracellular signaling domain comprising an intracellular domain of a costimulatory molecule and the combination of an ITAM containing primary cytoplasmic signaling sequence and a second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule; and/or

iii) between the ITAM containing primary cytoplasmic signaling sequence and the second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule.

4. The polypeptide of claim 1 , in combination with a protease inhibitor bound to the repressible protease, wherein the protease inhibitor is selected from grazoprevir (GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

5. The polypeptide of claim 4 , wherein the polypeptide is cleaved when a protease inhibitor is not bound to the repressible protease; and the polypeptide is not cleaved when the protease inhibitor is bound to the repressible protease.

6. The polypeptide of claim 1 , comprising:

a) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) a first intracellular signaling domain comprising an intracellular domain of a costimulatory molecule;

iv) a first protease cleavage site;

v) a repressible protease;

vi) a second protease cleavage site; and

vii) a second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule; and

viii) an ITAM containing primary cytoplasmic signaling sequence;

b) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) a first intracellular signaling domain comprising an intracellular domain of a costimulatory molecule;

iv) a first protease cleavage site;

v) a repressible protease;

vi) a second protease cleavage site;

vii) an ITAM containing primary cytoplasmic signaling sequence; and

viii) a second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule;

and/or

c) a polypeptide comprising SEQ ID NO: 35 or a sequence that is at least 70% identical to SEQ ID NO: 35 that maintains the same function.

7. The polypeptide of claim 1 , wherein the antigen bound by the antigen-binding fragment of an antibody is a tumor antigen.

8. The polypeptide of claim 1 , wherein the first and second intracellular signaling domain comprising an intracellular domain of a costimulatory molecule are, independently, a signaling domain from a protein selected from the group consisting of: CD2; CD7; CD27; CD28; CD30; CD40; CD 134 (OX40); CD137 (4-1 BB); CD273 (PD-L2); CD274 (PD-L1); CD278 (ICOS); LFA-1; LIGHT; NKG2C; and B7-H3.

9. The polypeptide of claim 1 , wherein the ITAM containing primary cytoplasmic signaling sequence is a signaling domain from a protein selected from the group consisting of: TCRC; FcRγ; FcRβ; CD3ζ; CD3γ; CD3δ; CD3ε; CD5; CD22; CD79a; CD79b; CARD11; CD54 (ICAM); CD83; CD150 (SLAMF1); CD152 (CTLA4); CD223 (LAG3); CD270 (HVEM); DAP10; LAT; KD2C SLP76 TRIM; ZAP70; and CD66d.

10. A system comprising:

a) a first polypeptide comprising a polypeptide of claim 1 ;

and

b) a second polypeptide comprising:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) at least one intracellular signaling domain; and

iv) a degron domain.

11. The system of claim 10 , wherein the first polypeptide further comprises a cofactor for the repressible protease and at least one protease cleavage site of the repressible protease.

12. The system of claim 11 , wherein:

a) the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3); and/or

b) the cofactor is an HSV NS4A domain.

13. The system of claim 10 , wherein the degron domain comprises a dihydrofolate reductase (DHFR) degron (DD) or a ligand-induced degradation (LID) domain.

14. The system of claim 10 , wherein the first polypeptide is in combination with:

a) a protease inhibitor bound to the repressible protease, wherein the protease inhibitor is selected from grazoprevir (GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir;

b) the second polypeptide is in combination with a degron stabilizer bound to the degron domain, wherein the degron is DHFR degron and the degron stabilizer is trimethoprim (TMP) or an analog thereof; and/or

c) the second polypeptide is in combination with a degron destabilizer bound to the degron domain, wherein the degron is a ligand-induced degradation (LID) domain and the degron destabilizer is Shield-1 or an analog thereof.

15. The system of claim 10 ,

wherein the second polypeptide comprises from the N-terminus to the C-terminus:

a) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) at least one intracellular signaling domain; and

iv) a degron domain: or

b) a polypeptide comprising SEO ID NO: 39 or a sequence that is at least 70% identical to SEQ ID NO: 39 that maintains the same function; and/or wherein the first polypeptide and second polypeptide are physically linked to one another and/or flank a self-cleaving peptide domain.

16. A system comprising:

a) a first polypeptide comprising:

i) extracellular binding domain;

ii) a transmembrane domain; and

iii) a peptide domain; and

b) a second polypeptide comprising a polypeptide of claim 1 .

17. The system of claim 16 , wherein the peptide domain is specifically bound by a repressible protease, optionally HCV NS3.

18. The system of claim 16 , wherein the second polypeptide further comprises a cofactor for the repressible protease; and/or wherein the first or second polypeptides do not comprise a protease cleavage site.

19. The system of claim 18 , wherein:

a) the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3);

b) the repressible protease is catalytically dead; and/or

c) the cofactor is an HSV NS4A domain.

20. The system of claim 16 , wherein the second polypeptide is in combination with a protease inhibitor bound to the repressible protease, wherein the protease inhibitor is selected from grazoprevir (GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

21. The system of claim 20 , wherein:

a) the repressible protease of the second polypeptide specifically binds the peptide domain of the first polypeptide in the absence of the protease inhibitor; and/or

b) the repressible protease of the second polypeptide does not specifically bind the peptide domain of the first polypeptide in the presence of the protease inhibitor.

22. The system of claim 16 , wherein the first polypeptide further comprises at least one intracellular signaling domain; and each of the at least one intracellular signaling domains of the first and/or second polypeptide independently comprises an intracellular signaling domain selected from the group consisting of: TCRC; FcRy; FcRp; CD3zeta; CD3y; CD35; CD3s; CD3C; CD22; CD79a; CD79b; CD66d; CARD11; CD2; CD7; CD27; CD28; CD30; CD40; CD54 (ICAM); CD83; CD134 (OX40); CD137 (4-1BB); CD150 (SLAMF1); CD152 (CTLA4); CD223 (LAG3); CD270 (HVEM); CD273 (PD-L2); CD274 (PD-L1); CD278 (ICOS); DAP10; LAT; KD2C SLP76; TRIM; ZAP70; and 41BB.

23. The system of claim 16 , wherein the first polypeptide comprises:

a) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain; and

iii) a peptide domain;

b) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) at least one intracellular signaling domain; and

iv) a peptide domain;

c) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) a single intracellular signaling domain; and

iv) a peptide domain;

d) a polypeptide comprising from the N-terminus to the C-terminus:

i) an extracellular binding domain;

ii) a transmembrane domain;

iii) a first intracellular signaling domain;

iv) a second intracellular signaling domain; and

v) a peptide domain; or

e) a polypeptide comprising SEQ ID NO: 84, SEQ ID NO: 85, or a sequence that at least 70% identical to SEQ ID NO: 84 or SEQ ID NO: 85 that maintains the same function.