Immunomodulatory fusion proteins and uses thereof
The present disclosure relates to immunomodulatory fusion proteins containing an extracellular binding domain and an intracellular signaling domain, wherein binding of a target can generate a modulatory signal in a host cell, such as a T cell. The present disclosure also relates to uses of immune cells expressing such immunomodulatory fusion proteins to treat certain diseases, such as cancer or infectious disease.
1. A fusion protein, comprising (a) an extracellular component comprising a binding domain that specifically binds a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component connecting the extracellular and intracellular components,
wherein the extracellular component is or comprises (i) a CD95 (Fas) ectodomain that binds to CD95L (FasL) or (ii) a CD95L (FasL)-binding fragment of (i), and the intracellular component is or comprises a CD137 (4-1BB) intracellular signaling domain or a signal-producing portion thereof.
2. The fusion protein according to claim 1 , wherein the extracellular component comprises a full length mature extracellular portion of a Fas protein.
3. A fusion protein, comprising (a) an extracellular component comprising a binding domain that specifically binds a target, (b) an intracellular component comprising an intracellular signaling domain, and (c) a hydrophobic component connecting the extracellular and intracellular components,
wherein the binding domain is, or has at least 95% identity to, an inhibitory molecule binding domain and the intracellular signaling domain is, or contains at least 95% identity to, a costimulatory or stimulatory molecule binding domain, and
wherein the inhibitory molecule is or comprises (i) a CD95 (Fas) ectodomain that binds to CD95L (FasL) or (ii) a CD95L (FasL)-binding fragment of (i), and the costimulatory or stimulatory molecule is or comprises an intracellular signaling domain or a signal-producing portion thereof from CD137 (4-1BB).
4. The fusion protein according to claim 1 , wherein the expression of the fusion protein in a T cell comprising an antigen-specific TCR or an antigen-specific chimeric antigen receptor results in at least about a 1.5-fold, 2-fold, or 3-fold increase in survival, expansion, cytotoxicity, and/or cytokine secretion by the T cell in response to the antigen, as compared to a response to the antigen by a cell substantially the same as the T cell but not containing the fusion protein.
5. The fusion protein according to claim 1 , wherein the extracellular component further comprises an additional extracellular portion, wherein (a) the additional extracellular portion optionally is from or shares identity with an extracellular portion of a molecule that is distinct from the binding domain source molecule or does not contain the binding domain or (b) the additional extracellular portion is from a hydrophobic component, or contains the hydrophobic component or a portion thereof.
6. The fusion protein according to claim 5 , wherein the extracellular component or additional extracellular portion comprises a multimerization domain or a spacer.
7. The fusion protein according to claim 6 , wherein the multimerization domain comprises an extracellular component modified to contain a cysteine residue within about 2 to about 15 amino acids from the hydrophobic component.
8. The fusion protein according to claim 1 , wherein the hydrophobic component comprises a transmembrane domain of a CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, or Zap70.
9. The fusion protein according to claim 1 , wherein (a) the extracellular component comprises the CD95 (Fas) ectodomain, (b) the hydrophobic component comprises a transmembrane domain of a CD137 (4-1BB), and (c) the intracellular component comprises the CD137 (4-1BB) intracellular signaling domain.
10. The fusion protein according to claim 1 , wherein (a) the extracellular component comprises an amino acid sequence encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NOs:71, 73, and 75, (b) the hydrophobic component comprises an amino acid sequence encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NOs.:4, 77, and 197, and (c) the intracellular component comprises the amino acid sequence encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NO.:13.
11. The fusion protein according to claim 10 , wherein the intracellular component comprises a second intracellular signaling domain.
12. The fusion protein according to claim 11 , wherein the second intracellular signaling domain is or comprises an intracellular signaling domain of a CD28.
13. The fusion protein according to claim 10 , wherein the extracellular component further comprises a multimerization domain with an amino acid sequence encoded by a nucleic acid molecule as set forth in SEQ ID NO.:9.
14. The fusion protein according to claim 1 , wherein (a) the extracellular component comprises an amino acid sequence selected from the group consisting of SEQ ID NOs.:72, 74, and 76 (b) the hydrophobic component comprises an amino acid sequence selected from the group consisting of SEQ ID NOs.:27, 78, and 198, and (c) the intracellular component comprises an amino acid sequence as set forth in SEQ ID NO.:36.
15. The fusion protein according to claim 1 , wherein the extracellular component further comprises a multimerization domain with an amino acid sequence as set forth in SEQ ID NO.:32.
16. A nucleic acid molecule encoding a fusion protein according to claim 1 .
17. A vector comprising a nucleic acid molecule according to claim 16 .
18. The vector according to claim 17 , further encoding an antigen-specific TCR.
19. A host cell, comprising a fusion protein according to claim 1 .
20. The fusion protein of claim 1 , wherein the extracellular portion of a complex formed by specific binding of the fusion protein to the target (fusion protein::target complex) is of a size, or spans a distance, of (i) up to about a distance between two cell membranes of an immunological synapse, (ii) up to about or substantially the same as a distance spanned by the extracellular portion of a complex between a T cell receptor (TCR) and an MHC-peptide complex specifically bound by the TCR, (iii) up to about or substantially the same as a distance spanned by the extracellular portion of a complex between a natural molecule comprising the binding domain and its cognate binding partner; (iii) less than or up to about 40 nm, 25 nm, 20 nm, 15 nm, or 14 nm; or (iv) any combination thereof.
21. The fusion protein of claim 1 , wherein (a) the extracellular component comprises the amino acid sequence set forth in SEQ ID NO: 72, (b) the hydrophobic component comprises the amino acid sequence set forth in SEQ ID NO: 78, and (c) the intracellular component comprises the amino acid sequence set forth in SEQ ID NO: 36.
22. The fusion protein of claim 1 , wherein the fusion protein comprises the amino acid sequence set for in SEQ ID NO: 186.