CHIMERIC TIM4 RECEPTORS AND USES THEREOF
The present disclosure relates to chimeric Tim4 receptors, host cells modified to include chimeric Tim4 receptor molecules, and methods of making and using such receptor molecules and modified cells.
1 . A polynucleotide encoding a chimeric Tim4 receptor comprising a single chain chimeric protein, the single chain chimeric protein comprising:
a receptor binding domain comprising a Tim4 binding domain;
an intracellular signaling domain comprising a CD28 costimulatory signaling domain and a CD3ζ signaling domain; and
a transmembrane domain positioned between and connecting the receptor binding domain and the intracellular signaling domain.
2 . The polynucleotide of claim 1 , wherein the Tim4 binding domain comprises the amino acid sequence of SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2.
3 . The polynucleotide of claim 1 , wherein the receptor binding domain further comprises an extracellular spacer domain positioned between the Tim4 binding domain and the transmembrane domain.
4 . The polynucleotide of claim 3 , wherein the extracellular spacer domain comprises an immunoglobulin hinge region, a hinge region of a type 1 membrane protein, a stalk region of a type II C-lectin, an immunoglobulin constant domain, or a fragment thereof.
5 . The polynucleotide of claim 4 , wherein the extracellular spacer domain comprises:
(a) an IgG1, IgG2, IgG3, IgG4, IgA, or IgD hinge region;
(b) a modified IgG4 hinge region comprising the amino acid sequence of SEQ ID NO: 3;
(c) a stalk region of a type II C-lectin selected from CD23, CD69, CD72, CD94, NKG2A, and NKG2D;
(d) a hinge region of a type 1 membrane protein selected from CD8a, CD4, CD28 and CD7; or
(e) an immunoglobulin constant region domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or any combination thereof.
6 . The polynucleotide of claim 1 , wherein the transmembrane domain comprises a Tim4, CD27, CD28, CD8, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3 transmembrane domain.
7 . The polynucleotide of claim 6 , wherein the transmembrane domain comprises a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO:28, a CD27 transmembrane domain comprising the amino acid sequence of SEQ ID NO:32, a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:29, a 4-1BB transmembrane domain comprising the amino acid sequence of SEQ ID NO:30, an OX40 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 31, a CD30 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, a CD40 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 37, a PD-1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 38, an ICOS transmembrane domain comprising the amino acid sequence of SEQ ID NO: 33, a LFA-1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:35, a CD2 transmembrane domain comprising the amino acid sequence of SEQ ID NO:34, or a CD7 transmembrane domain comprising the amino acid sequence of SEQ ID NO:39, a LIGHT transmembrane domain comprising the amino acid sequence of SEQ ID NO:40, a NKG2C transmembrane domain comprising the amino acid sequence of SEQ ID NO:41, or a B7-H3 transmembrane domain comprising the amino acid sequence of SEQ ID NO:42.
8 . The polynucleotide of claim 1 , wherein the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:4 or 62.
9 . The polynucleotide of claim 1 , wherein the transmembrane domain is a CD28 transmembrane domain.
10 . The polynucleotide of claim 1 , wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:63 or 19.
11 . The polynucleotide of claim 1 , wherein the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID NO:69 or amino acids 25-495 of SEQ ID NO:69.
12 . The polynucleotide of claim 1 , wherein the polynucleotide is codon optimized for expression in a host cell.
13 . An expression vector comprising a polynucleotide of claim 1 .
14 . The expression vector of claim 13 , wherein the expression vector is a viral vector.
15 . The expression vector of claim 13 , wherein the expression vector is a lentiviral vector.
16 . A modified T cell comprising an expression vector according to claim 13 .
17 . The modified T cell of claim 16 , wherein the T cell is a CD4+ T cell.
18 . The modified T cell of claim 16 , wherein the T cell is a CD8+ T cell.
19 . A pharmaceutical composition comprising a modified T cell according to claim 16 , and a pharmaceutically acceptable carrier, diluent, or excipient.