Targeted IL-12 heterodimeric Fc-fusion proteins

1. A targeted IL-12 heterodimeric Fc fusion protein comprising:

a) a first monomer comprising, from N- to C-terminal:

i) a first IL-12 protein domain;

ii) a first domain linker;

iii) a second IL-12 protein domain;

iv) a second domain linker; and

v) a first variant human IgG Fc domain comprising CH2-CH3; and

b) second monomer comprising, from N- to C-terminal:

i) an scFv domain;

ii) a third domain linker; and

iii) a second variant human IgG Fc domain comprising CH2-CH3;

wherein either said first IL-12 protein domain comprises an 1L-12p35 subunit and said second IL-12 protein domain comprises an 1L-12p40 subunit, or said first IL-12 protein domain comprises an 1L-12p40 subunit and said second IL-12 protein domain comprises an 1L-12p35 subunit,

wherein said first monomer binds a dimeric IL-12 receptor complex,

wherein said scFv domain comprises a variable heavy domain, an scFv linker, a variable light domain, and said scFv domain binds a target antigen, and

wherein said first and said second variant human IgG Fc domains comprise modifications promoting heterodimerization of said first and said second variant human IgG Fe domains, and

wherein said 1L-12p40 subunit is a variant 1L-12p40 subunit, and

wherein said variant 1L-12p40 subunit comprises an amino acid sequence that differs from that of SEQ ID NO:4 by virtue of an amino acid substitution at one or more amino acid residues selected from the group consisting of E59, K99, D18, K264, C252, N200, E3, D7, E12, D14, W15, P17, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, 5183, P185, E187, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, Q256, K258, K260, E262, D265, D270, N281, 0289, D290, R291, Y292, Y293, and E299.

2. The targeted IL-12 heterodimeric Fc fusion protein according to claim 1 , wherein said scFv domain binds a target antigen selected from the group consisting of human PD-1 and human PD-L1.

3. The targeted IL-12 heterodimeric Fc fusion protein according to claim 2 , wherein said scFv domain binds human PD-1 and does not compete for binding to said human PD-1 with nivolumab and/or pembrolizumab.

4. The targeted IL-12 heterodimeric Fc fusion protein according to claim 1 , wherein said IL-12p35 subunit is a variant IL-12p35 subunit, and

wherein said variant IL-12p35 subunit comprises an amino acid sequence that differs from that of SEQ ID NO:2 by virtue of an amino acid substitution at one or more amino acid residues selected from the group consisting of Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

5. The targeted IL-12 heterodimeric Fc fusion protein according to claim 1 , wherein said modifications promoting heterodimerization of said first and said second variant human IgG Fc domains are a set of amino acid substitutions selected from the group consisting of L368D/K370S and S364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S and S364K/E357Q; T366S/L368A/Y407V and T366W; and T366S/L368A/Y407V/Y349C and T366W/S354C, according to EU, numbering and/or wherein said first and said second variant human IgG Fc domains each comprise amino acid substitutions M428L/N434S, according to EU numbering.

6. One or more nucleic acids encoding a targeted IL-12 heterodimeric Fc fusion protein according to claim 1 .

7. A host cell comprising said one or more nucleic acids of claim 6 .

8. A method of making a targeted IL-12 heterodimeric Fc fusion protein, said method comprising culturing a host cell according to claim 7 under conditions whereby said targeted IL-12 heterodimeric Fc fusion protein is produced.

9. A targeted IL-12 heterodimeric Fc fusion protein comprising:

a) a first monomer comprising, from N- to C-terminal:

i) a first IL-12 protein domain;

ii) a first domain linker;

iii) a second IL-12 protein domain;

iv) a first variant human IgG Fc domain;

b) a second monomer comprising an antibody heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant human IgG Fc domain; and

c) a third monomer comprising an antibody light chain comprising VL-CL;

wherein said VH and VL domains form an antigen binding domain that binds a target antigen;

wherein either said first IL-12 protein domain comprises an IL-12p35 subunit and said second IL-12 protein domain comprises an IL-12p40 subunit, or said first IL-12 protein domain comprises an IL-12p40 subunit and said second IL-12 protein domain comprises an IL-12p35 subunit,

wherein said first monomer binds a dimeric IL-12 receptor complex,

wherein said first and said second variant human IgG Fc domains comprise modification promoting heterodimerization of said first and second variant human IgG Fe domains, and

wherein said IL-12p40 subunit is a variant IL-12p40 subunit, and

wherein said variant IL-12p40 subunit comprises an amino acid sequence that differs from that of SEQ ID NO:4 by virtue of an amino acid substitution at one or more amino acid residues selected from the group consisting of E59, K99, D18, K264, C252, N200, E3, D7, E12, D14, W15, P17, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, S204, F206, R208, D209, D214, N218, 0220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, Q256, K258, K260, E262, D265, D270, N281, 0289, D290, R291, Y292, Y293, and E299.

10. The targeted IL-12 heterodimeric Fc fusion protein according to claim 9 , wherein said antigen binding domain binds a target antigen selected from the group consisting of human PD-1 and human PD-L1.

11. The targeted IL-12 heterodimeric Fc fusion protein according to claim 9 , wherein said antigen binding domain binds human PD-1 and does not compete for binding to said human PD-1 with nivolumab and/or pembrolizumab.

12. The targeted IL-12 heterodimeric Fc fusion protein according to claim 9 , wherein said IL-12p35 subunit is a variant IL-12p35 subunit, and

wherein said variant IL-12p35 subunit comprises an amino acid sequence that differs from that of SEQ ID NO:2 by virtue of an amino acid substitution at one or more amino acid residues selected from the group consisting of Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

13. The targeted IL-12 heterodimeric Fe fusion protein according to claim 9 , wherein said modifications promoting heterodimerization of said first and said second variant human IgG Fe domains are a set of amino acid substitutions selected from the group consisting of L368D/K370S and S364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S and S364K/E357Q; and T366S/L368A/Y407V and T366W, T366S/L368A/Y407V/Y349C and T366W/S354C, according to EU numbering, and/or wherein said first and said second variant human IgG Fc domains each comprise amino acid substitutions M428L/N434S, according to EU numbering.

14. One or more nucleic acids encoding a targeted IL-12 heterodimeric Fc fusion protein according to claim 9 .

15. A host cell comprising said one or more nucleic acids of claim 14 .

16. A method of making a targeted IL-12 heterodimeric Fc fusion protein, said method comprising culturing a host cell according to claim 15 under conditions whereby said targeted IL-12 heterodimeric Fc fusion protein is produced.