Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains

1. A bifunctional heterodimeric protein comprising:

a) an IL-15/IL-15Rα fusion protein comprising a human IL-15Rα protein having the amino acid sequence of SEQ ID NO:4, a human IL-15 variant protein having the amino acid sequence of SEQ ID NO:2 and comprising one or more amino acid substitutions, and a first Fc domain,

wherein said human IL-15Rα protein is covalently attached to the N-terminus of said human IL-15 variant protein using a first domain linker and said human IL-15 variant protein is covalently attached to the N-terminus of said first Fc domain using a second domain linker, or

wherein said human IL-15 variant protein is covalently attached to the N-terminus of said human IL-15Rα protein using a first domain linker and said human IL-15Rα protein is covalently attached to the N-terminus of said first Fc domain using a second domain linker; and

b) an antigen binding domain monomer comprising a heavy chain comprising a VH-CH1-hinge-CH2-CH3 monomer, wherein VH is a variable heavy chain and CH2-CH3 is a second Fc domain, and a light chain comprising a variable light chain (VL) and a light constant domain (CL),

wherein said first and said second Fc domains are variants of a human IgG1 Fc domain and have a set of amino acid substitutions selected from the group consisting of S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q: L368D/K370S; L368D/K370S:S 364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K; L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EU numbering, and

wherein said antigen binding domain monomer binds human CD8.

2. The bifunctional heterodimeric protein according to claim 1 , wherein said first and/or said second Fc domains are variants of a human IgG1 Fc domain and have an additional set of amino acid substitutions comprising Q295E/N384D/Q418E/N421D, according to EU numbering.

3. The bifunctional heterodimeric protein according to claim 1 , wherein said first and/or said second Fc domains are variants of a human IgG1 Fc domain and have an additional set of amino acid substitutions consisting of G236R/L328R, E233P/L234V/L235A/G236de1/S239K, E233P/L234V/L235A/G236de1/S267K, E233P/L234V/L235A/G236de1/S239K/A327G, E233P/L234V/L235A/G236de1/S267K/A327G and E233P/L234V/L235A/G236del, according to EU numbering.

4. The bifunctional heterodimeric protein according to claim 1 , wherein said human IL-15 variant protein and said human IL-15Rα protein have a set of amino acid substitutions selected from the group consisting of E87C:D96/P97/C98; E87C:D 96/C97/A98; V49C:S40C; L52C:S40C; E89C:K34C; Q48C:G38C; E53C:L42C; C42S:A 37C; and L45C:A37C, respectively.

5. The bifunctional heterodimeric protein according to claim 1 , wherein said human IL-15 variant protein has one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D3ON, D61N, E64Q, N65D, and Q108E.

6. The bifunctional heterodimeric protein according to claim 1 , wherein said human IL-15 variant protein comprises the amino acid substitutions N4D/N65D or D3ON/E64Q/N65D.

7. The bifunctional heterodimeric protein according to claim 1 , wherein said bifunctional heterodimeric protein is any one selected from the group consisting of (a) SEQ ID NOS: 626, 630 and 635; (b) SEQ ID NOS: 640, 644 and 649; (c) SEQ ID NOS: 746, 750 and 755; (d) SEQ ID NOS: 801, 805 and 810; (e) SEQ ID NOS: 815, 819 and 824; (f) SEQ ID NOS: 829, 833 and 838; (g) SEQ ID NOS: 925, 929 and 934; (h) SEQ ID NOS: 939, 943 and 948; (i) SEQ ID NOS: 953, 957 and 962; (j) SEQ ID NOS: 967, 971 and 976; and (k) SEQ ID NOS: 981, 985 and 990.

8. A nucleic acid composition comprising:

a) a first nucleic acid encoding said IL-15/IL-15Rα fusion protein of claim 1 ; and

b) a second nucleic acid encoding said antigen binding domain monomer of claim 1 .

9. An expression vector composition comprising:

a) a first expression vector comprising said first nucleic acid of claim 8 ; and

b) a second expression vector comprising said second nucleic acid of claim 8 .

10. A host cell comprising said expression vector composition of claim 9 .

11. A method of making a bifunctional heterodimeric protein comprising culturing the host cell of claim 10 under conditions wherein said bifunctional heterodimeric protein is expressed, and recovering said heterodimeric protein.

12. A bifunctional heterodimeric protein selected from the group consisting of (a) SEQ ID NOS: 626, 630 and 635; (b) SEQ ID NOS: 640, 644 and 649; (c) SEQ ID NOS: 746, 750 and 755; (d) SEQ ID NOS: 801, 805 and 810; (e) SEQ ID NOS: 815, 819 and 824; (f) SEQ ID NOS: 829, 833 and 838; (g) SEQ ID NOS: 925, 929 and 934; (h) SEQ ID NOS: 939, 943 and 948; (i) SEQ ID NOS: 953, 957 and 962; (j) SEQ ID NOS: 967, 971 and 976; and (k) SEQ ID NOS: 981, 985 and 990.

13. A method of treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of the bifunctional heterodimeric protein of claim 12 to said patient.

14. An expression vector composition comprising one or more expression vectors each comprising a nucleic acid such that the one or more expression vectors encode a bifunctional heterodimeric protein selected from the group consisting of (a) SEQ ID NOS: 626, 630 and 635; (b) SEQ ID NOS: 640, 644 and 649; (c) SEQ ID NOS: 746, 750 and 755; (d) SEQ ID NOS: 801, 805 and 810; (e) SEQ ID NOS: 815, 819 and 824; (f) SEQ ID NOS: 829, 833 and 838; (g) SEQ ID NOS: 925, 929 and 934; (h) SEQ ID NOS: 939, 943 and 948; (i) SEQ ID NOS: 953, 957 and 962; (j) SEQ ID NOS: 967, 971 and 976; and (k) SEQ ID NOS: 981, 985 and 990.

15. A host cell comprising the expression vector composition of claim 14 .

16. A method of producing a bifunctional heterodimeric protein comprising (a) culturing the host cell of claim 15 under suitable conditions wherein said bifunctional heterodimeric protein is expressed, and (b) recovering said protein.