Modified granulocyte colony-stimulating factor (g-csf) and chimeric cytokine receptors binding same

A variant Granulocyte Colony-Stimulating Factor (G-CSF), wherein

• the variant G-CSF comprises at least one mutation in a site II interface region, at least one mutation in a site III interface region, or a combination thereof;
• wherein the at least one mutation in the site II interface region comprises at least one of:
• L108K, D112R, E122R E122K, E123K, and E123R relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO. 1;
• wherein the at least one mutation in the site III interface region comprises mutation E46R relative to the corresponding amino acid positions of the sequence shown in SEQ ID NO. 1;
• and wherein the variant G-CSF binds selectively to a receptor comprising a variant extracellular domain (ECD) of Granulocyte Colony-Stimulating Factor Receptor (G-CSFR).

A chimeric receptor, comprising:

• (a) an ECD operatively linked to at least one second domain, the second domain comprising:
• (b) an ICD comprising at least one signaling molecule binding site from an intracellular domain of a cytokine receptor;
• wherein
• the at least one signaling molecule binding site is selected from the group consisting of:
• a SHC binding site of Interleukin (IL)-2Rβ;
• a STAT5 binding site of IL-2Rβ, an IRS-1 or IRS-2 binding site of IL-4Rα, a STAT6 binding site of IL-4Rα, a SHP-2 binding site of glycoprotein 130 (gp130), a STAT3 binding site of gp130, a SHP-1 or SHP-2 binding site of Erythropoietin Receptor (EPOR), a STAT5 binding site of EPOR, a STAT1 or STAT2 binding site of Interferon Alpha And Beta Receptor Subunit 2 (IFNAR2), and a STAT1 binding site of Interferon Gamma Receptor 1 (IFNγR1), or combinations thereof;
• wherein
• the ICD further comprises at least one Box 1 region and at least one Box 2 region of at least one protein selected from the group consisting of G-CSFR, gp130, EPOR, and Interferon Gamma Receptor 2 (IFNγR2), or combinations thereof;
• and
• (c) at least one third domain comprising a TMD;
• wherein the ECD is N-terminal to the TMD, and the TMD is N-terminal to the ICD.

A system for selective activation of a cell, optionally an immune cell, the system comprising:

• (i) a receptor comprising a variant ECD of G-CSFR;
• (ii) a variant G-CSF that selectively binds the receptor of (i);
• and
• (iii) one or both of:
• (a) at least one additional agonistic or antagonistic signaling protein;
• and
• (b) at least one antigen binding signaling receptor;
• wherein the variant ECD of G-CSFR comprises at least one mutation in a site II interface region, at least one mutation in a site III interface region, or combinations thereof;
• wherein
• i) the at least one mutation in the site II interface region is located at an amino acid position of the G-CSFR ECD selected from the group consisting of amino acid position 141, 167, 168, 171, 172, 173, 174, 197, 199, 200, 202 and 288 of the sequence shown in SEQ ID NO. 2;
• ii) the at least one mutation in the site II interface region of the G-CSFR ECD is selected from the group consisting of R141E, R167D, K168D, K168E, L171E, L172E, Y173K, Q174E, D197K, D197R, M199D, D200K, D200R, V202D, R288D, and R288E of the sequence shown in SEQ ID NO. 2;
• iii) the at least one mutation in the site III interface region of the G-CSFR ECD is selected from the group consisting of amino acid position 30, 41, 73, 75, 79, 86, 87, 88, 89, 91, and 93 of amino acids 2-308 of the sequence shown in SEQ ID NO. 2;
• or
• iv) the at least one mutation in the site III interface region of the G-CSFR ECD is selected from the group consisting of S30D, R41E, Q73W, F75KF, S79D, L86D, Q87D, I88E, L89A, Q91D, Q91K, and E93K of the sequence shown in SEQ ID NO. 2;
• or
• wherein the G-CSFR ECD comprises the mutations:
• R41E, R141E, and R167D of the sequence shown in SEQ ID NO. 2.

A chimeric receptor, comprising:

• (i) an ECD of Interleukin Receptor alpha (IL-7Rα);
• (ii) a TMD;
• and
• (iii) an ICD of a cytokine receptor that is distinct from a wild-type, human IL-7Rα intracellular signaling domain set forth in SEQ ID NO:109;
• wherein the ECD and TMD are each operatively linked to the ICD.