Chimeric endocytic receptors and method of use thereof
The invention discloses chimeric endocytic receptor CER-based constructs for activating and regulating immune response, and method for using the same. The CER-based constructs are based on the structure of FcγRI/γ chain and incorporate high-affinity binding domain from receptors or antibodies shown to uptake specific antigen and present the antigen to T cells or B cells to initiate the antigen-specific immune response, Such design has the ability to transform native monocytes or T cells to CER-expressing monocytes (CER-M) or CER-expressing T cells (CER-T) in recognizing and uptake the target antigen and activate subsequent immune responses. Such engineered CER-M or CER-T can be used to treat tumor, viral diseases and autoimmune diseases directly. The endocytosis process with involvement of FcR-γ may enhance and coordinate T cell activation in combination with T cell activation by other types of constructs such as CAR.
1. A chimeric endocytic antigen receptor (CER) complex comprising:
a) a homodimer of a chimeric polypeptide comprising an engagement element and a human Fc receptor transmembrane and cytoplasmic domain, and
b) two homodimers of Fc receptor gamma chain (FcR-γ),
wherein each chimeric polypeptide is associated with one of the homodimers of FcR-γ at the human Fc receptor transmembrane and cytoplasmic domain of the chimeric polypeptide.
2. The complex of claim 1 , wherein the engagement element is an antibody fragment with high affinity to a target antigen, or an extracellular domain of a receptor.
3. The complex of claim 2 , wherein the target antigen is virus associated antigens (VA) selected from the group consisting of: HPV associated antigen E6/E7, HBV antigen HBs Ag/HBe Ag, EBV antigens EBNA1/LMP1/LMP2/EBER, CMV antigen pp65/pp150/pp52/, HIV antigen p24, RSV, influenza A and B viruses, parainfluenza viruses, adenoviruses, and coronavirus associated antigen S1/S2/N.
4. The complex of claim 2 , wherein the target antigen is selected from the group consisting of:
mutated CDK4, CTNNB1, CASP8, P53, KRAS, NRAS, EGFR, EGFRVIII, BRCA1, BRCA2, PALB2, ATM, RAD51D, RECQL, CHEK2, c-MET,
melanoma-antigen encoding (MAGE), MAGEA/MAGEB/MAGEC, BAGE, GAGE, LAGE/NY-ESO1, SSX,
tyrosinase, gp100/pmel17, Melan-A/MART-1, gp75/TRP1, TRP2, CEA, CLL1, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66ae, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, gp100,
RAGE-1, PRAME, survivin, ERBB2 (HER2/NEU), protein Wilms tumor 1 (WT1), EpCAM, MUC1 (CA15-3), MUC2, MUC3, MUC4, MUC6, MUC16, PMSA, Placental growth factor (PIGF), HIF-1a, EGP-1 (TROP-2), EGP-2, survivin, epidermal glycoprotein 1 (EGP-1, TROP2), EGP-2, FLT3, G250, folate receptor, GAGE, gp100, HLA-DR, CD317 (HM1.24), HMGB-1,
CEA (CEACAM-5), CEACAM-6, AFP, OCT4, CD133, CD90, CD13, c-MET, CDC27,
CXCR2, CXCR4, CXCR7, CCR5, CCR7, CCR9, CCR10, CX3CR1, PD-L1, VISTA, and Siglec-15.
5. The complex of claim 1 , wherein the engagement element is an extracellular domain of a ligand, or a self-antigen.
6. The complex of claim 5 , wherein the ligand or the self-antigen is selected from the group consisting of insulin, Glutamic Acid Decarboxylase (GAD), and insulinoma-associated protein 2 (IA2).
7. The complex of claim 1 , wherein the chimeric polypeptide further comprises a hinge between the engagement element and the human Fc receptor transmembrane and cytoplasmic domain, wherein the hinge is selected from the group consisting of IgG1 hinge, IgG2 hinge, IgG3 hinge, and IgG4 hinge.
8. The complex of claim 1 , wherein the human Fc receptor transmembrane and cytoplasmic domain comprises a transmembrane domain and a cytoplasmic domain of an immunoglobulin Fc receptor selected from the group consisting of: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).
9. The complex of claim 8 , wherein the Fc receptor transmembrane and cytoplasmic domain comprises FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 5 and FcγRI cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 6.
10. The complex of claim 1 , wherein the FcR-γ comprises the amino acid sequence of SEQ ID NO:11.
11. An engineered T cell expressing the complex of claim 1 , wherein the cell further expresses CD3+, CD8+, or CD4+ T cell markers.
12. The complex of claim 1 , further comprising a costimulatory ligand selected from the group consisting of: CD80, CD86, or CD40L.
13. The engineered T cell of claim 11 , further comprising a Chimeric Antigen Receptor (CAR).
14. The engineered T cell of claim 13 , wherein the chimeric antigen receptor (CAR) further comprises heavy chain variable region (VH) and light chain variable region (VL) portions of a single-chain fragment variable (scFv) that targets a tumor associated antigen (TAA); a hinge of a CD8a hinge or IgG4 hinge; and an intracellular effector consisting of one or more co-stimulatory signaling domains comprising CD28 intracellular domains (endodomain) or 4-1BB intracellular domain.
15. An engineered human monocyte expressing the complex of claim 1 , wherein the cell is derived from a donor or an autologous monocyte.
16. A chimeric endocytic antigen receptor (CER) polypeptide comprising a chimeric polypeptide comprising an engagement element, and a human Fc receptor transmembrane and cytoplasmic domain, wherein the human Fc receptor transmembrane and cytoplasmic domain is linked to a Fc receptor gamma chain (FcR-γ) via a self-cleavage peptide.
17. The CER polypeptide of claim 16 , wherein the self-cleavage peptide is selected from the group consisting of: T2A, P2A, E2A, and F2Z.