IL-12 variants, anti-PD1 antibodies, fusion proteins and uses thereof

Abstract

An IL-12 variant is provided. Antibodies that specifically bind to PD1 are also provided. A fusion protein of an IL-12 variant and an anti-PD1 antibody is also provided. Uses of these IL-12 variants, anti-PD1 antibodies, fusion proteins, and related compositions and methods are also provided.

Description

Background of the Invention

[0002] The present invention relates to interleukin 12 (IL-12) variants, and methods of preparing and using the variants. The present invention also provides anti-PD1 antibodies, and fusion proteins comprising such IL-12 variants and anti-PD1 antibodies. The present invention also relates to related molecules, such as nucleic acids encoding such IL-12 variants, fusion proteins and antibodies, and related compositions and methods.

[0003] Interleukin 12 (IL-12) is a cytokine with multiple functions in the immune system. IL-12 is a heterodimer containing two subunits, p35 (encoded by the IL-12A gene) and p40 (encoded by the IL-12B gene). IL-12 binds to and cross-links the heterodimeric IL-12 receptor (IL-12R) chains IL-12Rβ1 and IL-12Rβ2. IL-12R is upregulated by T cell receptor (TCR) activation, thereby enhancing the sensitivity of T cells to IL-12 stimulation. After IL-12 binds to IL-12R, STAT4 is phosphorylated (pSTAT4) and pSTAT4 promotes IL-12-dependent effects, including interferon gamma (IFNγ) production and the cytolytic capacity of CD8 T cells, CD4 T cells, T regulatory cells, and NK cells.

[0004] Preclinical models suggest that IL-12 promotes antitumor immunity by acting directly on T cells and NK cells and indirectly on antigen-presenting cells in the tumor microenvironment (TME). However, preclinical and clinical studies also suggest that IL-12R agonists may have dose-limiting toxicities. These studies speculate that systemic activity may result in significant toxicity.

[0005] Programmed cell death protein 1 (PD1) is an important cell surface receptor that inhibits T cell activation signals and functions as a checkpoint molecule to limit anti-tumor immunity. Although some PD1 expression has been observed in multiple immune cell subsets including B cells and innate immune cells, high PD1 expression is mainly seen on CD8 and CD4 tumor-infiltrating lymphocytes (TILs) and is enriched in the TME compared with circulating T cell subsets.

[0006] Previous studies have targeted cytokines including IL-15, IL-12, and IFN to PD1-positive cells by fusing anti-PD1 antibodies with cytokine mutant protein variants (partial agonists). (Xu, Y., et al., Cancer Immunol Res, 2021. 9(10): p.1141-1157; Codarri Deak, L., et al., Nature, 2022. 610(7930): p.161-172; Hashimoto, M., et al., Nature, 2022. 610(7930): p.173-181. Garcin, G., et al., Nat Commun, 2014. 5: p.3016.).

[0007] However, there remains a need for improved IL-12 variants and related fusion proteins. SUMMARY OF THE INVENTION

[0009] The present disclosure provides interleukin 12 (IL-12) variants and methods for preparing and using the variants. The present invention also provides anti-PD1 antibodies and fusion proteins comprising IL-12 variants and anti-PD1 antibodies. In some embodiments, the IL-12 variants provided herein have reduced activity compared to wild-type IL-12. In some embodiments, the anti-PD1 antibodies provided herein can bind to PD1 while PD1 binds to PDL1 (e.g., anti-PD1 antibodies bind to different positions on PD1 that bind to PDL1). In some embodiments, the IL-12 variants / anti-PD1 fusion proteins provided herein have activity that is biased toward the tumor microenvironment (TME), rather than systemic activity. In some embodiments, the IL-12 variants / anti-PD1 fusion proteins provided herein have an improved therapeutic index compared to wild-type IL-12 and its fusion proteins.

[0010] The present disclosure further encompasses the expression of IL-12 variants, anti-PD1 antibodies and fusion proteins, as well as the preparation and production of compositions comprising the IL-12 variants, antibodies and fusion proteins of the present disclosure, such as medicaments using the IL-12 variants, antibodies and fusion proteins.

[0011] In some embodiments, provided herein are isolated human interleukin 12 (IL-12) variants comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and position D93 of SEQ ID NO: 2 (IL-12p40 subunit).

[0012] In some embodiments, provided herein is an isolated human interleukin 12 (IL-12) variant comprising the amino acid substitution Y167A of SEQ ID NO: 1 (IL-12p35 subunit) and the amino acid substitution D93L of SEQ ID NO: 2 (IL-12p40 subunit).

[0013] In some embodiments, provided herein are isolated human interleukin 12 (IL-12) variants comprising one or both of i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (variant IL-12p35 subunit) and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (variant IL-12p40 subunit).

[0014] In some embodiments, provided herein is an isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at one or more of the following positions: F39 of SEQ ID NO: 1 (IL-12p35 subunit), I52 of SEQ ID NO: 1, Y167 of SEQ ID NO: 1, K85 of SEQ ID NO: 2 (IL-12p40 subunit), and D93 of SEQ ID NO: 2.

[0015] In some embodiments, provided herein is an isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 10 or 37, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 11, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 14.

[0016] In some embodiments, provided herein is an isolated antibody that binds to PD1, comprising: a VH amino acid sequence comprising VH CDR1, VH CDR2, and VH CDR3 of the amino acid sequence of SEQ ID NO:7, and VL CDR1, VL CDR2, and VL CDR3 of the amino acid sequence of SEQ ID NO:8.

[0017] In some embodiments, provided herein is an isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:5, 51 or 52 and a light chain comprising the amino acid sequence of SEQ ID NO:6, wherein the C-terminal lysine of SEQ ID NO:5, 51 or 52 is optionally present.

[0018] In some embodiments, provided herein is an isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 19, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 20 or 37, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 22, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 23, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0019] In some embodiments, provided herein is an isolated antibody that binds to PD1, comprising: a VH amino acid sequence comprising VH CDR1, VH CDR2, and VH CDR3 of the amino acid sequence of SEQ ID NO: 17, and VL CDR1, VL CDR2, and VL CDR3 of the amino acid sequence of SEQ ID NO: 18.

[0020] In some embodiments, provided herein is an isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, 53 or 54 and a light chain comprising the amino acid sequence of SEQ ID NO: 16, wherein the C-terminal lysine of SEQ ID NO: 15, 53 or 54 is optionally present.

[0021] In some embodiments, provided herein is an isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 38 or 39, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 40, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 41.

[0022] In some embodiments, provided herein is an isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, wherein the fusion protein comprises a polypeptide of SEQ ID NO: 5, 25, 6, and 4.

[0023] In some embodiments, provided herein is an isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, wherein the fusion protein comprises the polypeptides of SEQ ID NOs: 15, 26, 16, and 4.

[0024] In some embodiments, provided herein are isolated polynucleotides comprising one or more nucleotide sequences encoding a human interleukin 12 (IL-12) variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and position D93 of SEQ ID NO: 2 (IL-12p40 subunit), wherein the one or more nucleotide sequences comprise the nucleotide sequence of SEQ ID NO: 44 and the nucleotide sequence of SEQ ID NO: 45.

[0025] In some embodiments, provided herein is one or more isolated polynucleotides comprising one or more nucleotide sequences encoding VH, VL, or both of an antibody that binds to PD1, wherein the polynucleotide comprises the VH nucleic acid sequence of SEQ ID NO: 46, the VL nucleic acid sequence of SEQ ID NO: 47, or both the VH nucleic acid sequence of SEQ ID NO: 46 and the VL nucleic acid sequence of SEQ ID NO: 47.

[0026] In some embodiments, provided herein is an isolated polynucleotide comprising one or more nucleotide sequences encoding any one or more of a heavy chain, a light chain, an IL-12p40 subunit, or a heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD1 antibody, wherein the polynucleotide comprises a heavy chain nucleic acid sequence of SEQ ID NO: 48, a light chain nucleic acid sequence of SEQ ID NO: 50, an IL-12p40 subunit nucleic acid sequence of SEQ ID NO: 45, a heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49, or each of a heavy chain nucleic acid sequence of SEQ ID NO: 48, a light chain nucleic acid sequence of SEQ ID NO: 50, an IL-12p40 subunit nucleic acid sequence of SEQ ID NO: 45, and a heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49.

[0027] In some embodiments, provided herein is an isolated polynucleotide comprising one or more nucleotide sequences encoding any one or more of a heavy chain, a light chain, an IL-12p40 subunit, or a heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD1 antibody, wherein the polynucleotide comprises a nucleic acid sequence encoding a heavy chain of an insert fragment of a plasmid deposited with ATCC having ATCC Accession No. PTA-127517, a nucleic acid sequence encoding a light chain of an insert fragment of a plasmid deposited with ATCC having ATCC Accession No. PTA-127519, a nucleic acid sequence encoding an IL-12p40 subunit of an insert fragment of a plasmid deposited with ATCC having ATCC Accession No. PTA-127520, a nucleic acid sequence encoding a heavy chain-IL12 p35 fusion polypeptide of an insert fragment of a plasmid deposited with ATCC having ATCC Accession No. PTA-127518. p35 fusion polypeptide, or the nucleic acid sequence encoding the heavy chain of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127517, the nucleic acid sequence encoding the light chain of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127519, the nucleic acid sequence encoding the IL-12p40 subunit of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127520, and the nucleic acid sequence encoding the heavy chain-IL12 p35 fusion polypeptide of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127518. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A and Figure 1B Comparative antibody GBT-PD1-0013 [in Figure 1A and 1B PD1(NB)] and known blocking anti-human PD1 antibodies [ Figure 1A and 1B The results of the determination of the ability of PD1 to bind to PD1. Figure 1A and 1B In FIG, the X-axis shows antibodies incubated with cells expressing PD1. Figure 1A In Figure 5, the Y axis shows the % of cells expressing PD1 bound by GBT-PD1-0013 / PD1(NB). Figure 1B In the figure, the Y axis shows the % of cells expressing PD1 that are bound by PD1 (B).

[0030] Figure 2 A schematic diagram showing the structure of the IL-12 mutant protein / anti-PD1 fusion protein provided herein is shown.

[0031] Figure 3The results of the assay comparing the activity of various mouse surrogate IL-12 mutant proteins / anti-PD1 fusion proteins are shown. The X-axis shows the concentration of IL-12 mutant proteins / anti-PD1 fusion proteins (ligands) (micrograms / ml), and the Y-axis shows the concentration of interferon gamma (IFNγ) (picograms / ml). DETAILED DESCRIPTION OF THE INVENTION

[0033] By reference to the following detailed description of embodiments of the present invention and the examples included herein, the present invention can be more easily understood. It should be understood that the present invention is not limited to specific production methods, which of course can vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0034] Example embodiments (E) of the present invention provided herein include:

[0035] E1. An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and at position D93 of SEQ ID NO: 2 (IL-12p40 subunit).

[0036] E2. An IL-12 variant of E1 wherein the Y167 substitution is Y167A

[0037] E3. The IL-12 variant of any one of E1 to E2, wherein the D93 substitution is D93L

[0038] E4. The IL-12 variant of any one of E1 to E3, wherein the Y167 substitution is Y167A and the D93 substitution is D93L.

[0039] E5. The IL-12 variant of any one of E1 to E4, wherein the p40 subunit further comprises one or more mutations to reduce the binding of IL-12 to heparin.

[0040] E6. The IL-12 variant of E5, wherein the mutations that reduce IL-12 binding to heparin comprise substitutions of K258G, S259G and K260G and deletions of R261, E262, K263 and K264 of SEQ ID NO:2.

[0041] E7. An isolated human interleukin 12 (IL-12) variant comprising the amino acid substitution Y167A of SEQ ID NO: 1 (IL-12p35 subunit) and the amino acid substitution D93L of SEQ ID NO: 2 (IL-12p40 subunit).

[0042] E8. The IL-12 variant of E7, wherein the p40 subunit further comprises one or more mutations to reduce the binding of IL-12 to heparin.

[0043] The IL-12 variant of E9.E8, wherein the mutations that reduce IL-12 binding to heparin comprise substitutions of K258G, S259G and K260G and deletions of R261, E262, K263 and K264 of SEQ ID NO:2.

[0044] E10. An isolated human interleukin 12 (IL-12) variant comprising one or both of i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (variant IL-12p35 subunit) and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (variant IL-12p40 subunit).

[0045] E11. The IL-12 variant of E10, wherein the IL-12 variant comprises i) a polypeptide comprising the amino acid sequence of SEQ ID NO:3 and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:4.

[0046] E12. An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at one or more of the following positions: F39 of SEQ ID NO:1 (IL-12p35 subunit), I52 of SEQ ID NO:1, Y167 of SEQ ID NO:1, K85 of SEQ ID NO:2 (IL-12p40 subunit), and D93 of SEQ ID NO:2.

[0047] E13. The IL-12 variant of E12, wherein the F39 substitution is F39R or F39A.

[0048] E14. The IL-12 variant of any one of E12 to E13, wherein the I52 substitution is I52E, I52R or I52H.

[0049] E15. The IL-12 variant of any one of E12 to E14, wherein the Y167 substitution is Y167A.

[0050] E16. The IL-12 variant of any one of E12 to E15, wherein the K85 substitution is K85E.

[0051] E17. The IL-12 variant of any one of E12 to E16, wherein the D93 substitution is D93L.

[0052] E18. The IL-12 variant of any one of E12 to E17, wherein the p40 subunit further comprises one or more mutations to reduce binding of IL-12 to heparin.

[0053] E19. The IL-12 variant of E18, wherein the mutations that reduce IL-12 binding to heparin comprise substitutions of K258G, S259G and K260G and deletions of R261, E262, K263 and K264 of SEQ ID NO:2.

[0054] E20. The IL-12 variant of any one of E1 to E19, wherein the IL-12 variant has one or both of the following: i) reduced binding to the human IL-12 receptor compared to wild-type human IL-12 binding to the human IL-12 receptor, and ii) reduced activity on the human IL-12 receptor compared to wild-type human IL-12.

[0055] E21. An isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO:9, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO:10 or 37, VH CDR3 comprises the amino acid sequence of SEQ ID NO:11, VL CDR1 comprises the amino acid sequence of SEQ ID NO:12, VL CDR2 comprises the amino acid sequence of SEQ ID NO:13, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:14.

[0056] E22. The antibody of E21, wherein VH comprises the amino acid sequence of SEQ ID NO:7 or a variant of SEQ ID NO:7 comprising 1 to 4 amino acid substitutions at residues not within the CDRs, and VL comprises the amino acid sequence of SEQ ID NO:8 or a variant of SEQ ID NO:8 comprising 1 to 4 amino acid substitutions at residues not within the CDRs.

[0057] E23. The antibody of E22, wherein VH comprises the amino acid sequence of SEQ ID NO:7 and VL comprises the amino acid sequence of SEQ ID NO:8.

[0058] E24. An isolated antibody that binds to PD1, comprising: a VH amino acid sequence comprising VHCDR1, VH CDR2 and VH CDR3 of the amino acid sequence of SEQ ID NO:7, and VL CDR1, VL CDR2 and VL CDR3 of the amino acid sequence of SEQ ID NO:8.

[0059] E25. An isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 5, 51 or 52, and the light chain comprises the amino acid sequence of SEQ ID NO: 6, wherein the C-terminal lysine of SEQ ID NO: 5, 51 or 52 is optionally present.

[0060] E26. An isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 19, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 20 or 37, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 22, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 23, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0061] E27.E26 antibody, wherein VH comprises the amino acid sequence of SEQ ID NO: 17 or a variant of SEQ ID NO: 17 comprising 1 to 4 amino acid substitutions at residues not within the CDRs, and VL comprises the amino acid sequence of SEQ ID NO: 18 or a variant of SEQ ID NO: 18 comprising 1 to 4 amino acid substitutions at residues not within the CDRs.

[0062] E28. The antibody of E27, wherein VH comprises the amino acid sequence of SEQ ID NO:17 and VL comprises the amino acid sequence of SEQ ID NO:18.

[0063] E29. An isolated antibody that binds to PD1, comprising: a VH amino acid sequence comprising VHCDR1, VH CDR2 and VH CDR3 of the amino acid sequence of SEQ ID NO: 17, and VL CDR1, VLCDR2 and VL CDR3 of the amino acid sequence of SEQ ID NO: 18.

[0064] E30. An isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 15, 53 or 54, and the light chain comprises the amino acid sequence of SEQ ID NO: 16, wherein the C-terminal lysine of SEQ ID NO: 15, 53 or 54 is optionally present.

[0065] E31. An isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 38 or 39, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 40, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 41.

[0066] E32. The antibody of E31, wherein VH comprises the amino acid sequence of SEQ ID NO:33 and VL comprises the amino acid sequence of SEQ ID NO:34.

[0067] E33. The antibody of any one of E21 to E32, wherein the antibody does not block the binding of PDL1 to PD1.

[0068] E34. The antibody of any one of E21 to E33, wherein the antibody does not block the binding of an anti-PD1 antibody that inhibits the interaction between PD1 and PDL1 to PD1.

[0069] E35. The antibody of any one of E21 to E34, wherein the antibody has a modification in the Fc domain to reduce binding to Fcγ receptors.

[0070] E36. An isolated fusion protein comprising the human interleukin 12 (IL-12) variant of any one of E1 to E20 linked to an anti-PD1 antibody.

[0071] The fusion protein of E37.E36, wherein the anti-PD1 antibody is the antibody of any one of E21 to E34.

[0072] E38. The fusion protein of any one of E36 to E37, wherein the IL-12 variant comprises an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and at position D93 of SEQ ID NO: 2 (IL-12p40 subunit).

[0073] A fusion protein of E39.E38, wherein the Y167 substitution is Y167A and the D93 substitution is D93L.

[0074] E40. The fusion protein of any one of E36 to E39, wherein the IL-12 variant comprises one or both of i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (variant IL-12p35 subunit) and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (IL-12p40 subunit).

[0075] E41. The fusion protein of any one of E36 to E40, wherein the IL-12 variant comprises i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0076] E42. The fusion protein of any one of E36 to E41, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid sequence of SEQ ID NO:8.

[0077] E43. The fusion protein of any one of E36 to E41, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO:17 and the VL comprises the amino acid sequence of SEQ ID NO:18.

[0078] E44. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, wherein the fusion protein comprises the polypeptides of SEQ ID NOs: 5, 25, 6 and 4.

[0079] E45. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PDl antibody, wherein the fusion protein comprises polypeptides of SEQ ID NOs: 15, 26, 16 and 4.

[0080] E46. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding one or more of the IL-12 variants, anti-PD1 antibodies, fusion proteins or polypeptides thereof of any one of E1 to E45.

[0081] E47. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding a human interleukin 12 (IL-12) variant, the variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and position D93 of SEQ ID NO: 2 (IL-12p40 subunit), wherein the one or more nucleotide sequences comprise the nucleotide sequence of SEQ ID NO: 44 and the nucleotide sequence of SEQ ID NO: 45.

[0082] E48. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding VH, VL, or both of an antibody that binds to PD1, wherein the polynucleotide comprises the VH nucleic acid sequence of SEQ ID NO: 46, the VL nucleic acid sequence of SEQ ID NO: 47, or both the VH nucleic acid sequence of SEQ ID NO: 46 and the VL nucleic acid sequence of SEQ ID NO: 47.

[0083] E49. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding any one or more of the heavy chain, light chain, IL-12p40 subunit or heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD1 antibody, wherein the polynucleotide comprises the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12p40 subunit nucleic acid sequence of SEQ ID NO: 45, the heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49, or each of the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12p40 subunit nucleic acid sequence of SEQ ID NO: 45, and the heavy chain-IL12p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49.

[0084] E50. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding any one or more of the heavy chain, light chain, IL-12p40 subunit, or heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD-1 antibody, wherein the polynucleotide comprises a nucleic acid sequence encoding a heavy chain as an insert of a plasmid deposited with ATCC having ATCC Accession No. PTA-127517, a nucleic acid sequence encoding a light chain as an insert of a plasmid deposited with ATCC having ATCC Accession No. PTA-127519, a nucleic acid sequence encoding an IL-12p40 subunit as an insert of a plasmid deposited with ATCC having ATCC Accession No. PTA-127520, a nucleic acid sequence encoding a heavy chain-IL12 p35 subunit as an insert of a plasmid deposited with ATCC having ATCC Accession No. PTA-127518, p35 fusion polypeptide, or the nucleic acid sequence encoding the heavy chain of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127517, the nucleic acid sequence encoding the light chain of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127519, the nucleic acid sequence encoding the IL-12p40 subunit of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127520, and the nucleic acid sequence encoding the heavy chain-IL12p35 fusion polypeptide of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127518.

[0085] E51. The polynucleotide of any one of E46 to E50, wherein the polynucleotide is RNA or DNA.

[0086] E52. The polynucleotide of any one of E46 to E51, wherein the polynucleotide comprises at least one chemical modification.

[0087] The polynucleotide of E53.E52, wherein the chemical modification is selected from pseudouridine, 1-methylpseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-azauridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0088] E54. The polynucleotide of any one of E46 to E53, wherein the polynucleotide does not comprise a chemical modification.

[0089] E55. A vector comprising the polynucleotide of any one of E46 to E54.

[0090] E56. An isolated host cell comprising the polynucleotide of any one of E46 to E54 or the vector of E55.

[0091] E57. A method for producing an IL-12 variant, an anti-PD1 antibody or a fusion protein, comprising culturing the host cell of E56 under conditions that lead to the production of the IL-12 variant, the anti-PD1 antibody or the fusion protein, and optionally further recovering the IL-12 variant, the anti-PD1 antibody or the fusion protein.

[0092] E58. A pharmaceutical composition comprising the IL-12 variant of any one of E1 to E45, an anti-PD1 antibody or fusion protein, and a pharmaceutically acceptable carrier.

[0093] E59. A method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of E58 or the IL-12 variant, anti-PD1 antibody, or fusion protein of any one of E1 to E45.

[0094] E60. The IL-12 variant, anti-PD1 antibody or fusion protein of any one of E1 to E45 for use as a medicament, optionally for use as a medicament for cancer.

[0095] E61. The IL-12 variant, anti-PD1 antibody or fusion protein of any one of E1 to E45 for use in treating cancer.

[0096] E62. The IL-12 variant, anti-PD1 antibody, fusion protein or method of any one of E59 to E61, wherein the cancer is bladder cancer, breast cancer, clear cell renal carcinoma, head / neck squamous cell carcinoma [head and neck squamous cell carcinoma (SCCHN)], lung squamous cell carcinoma, lung adenocarcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma (RCC), small cell lung cancer (SCLC), triple-negative breast cancer, urothelial carcinoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL), endometrial cancer, B-cell acute lymphoma lymphoblastic leukemia, colorectal cancer (CRC), glioblastoma, uterine cancer, cervical cancer, penile cancer, gastric cancer (GC), non-melanoma skin cancer, NSCLC previously treated with platinum therapy and / or checkpoint inhibitors (e.g., PD(L)1 inhibitors), RCC previously treated with tyrosine kinase inhibitors and / or checkpoint inhibitors (e.g., PD(L)1 inhibitors), ovarian cancer, microsatellite stable (MSS) CRC, hepatocellular carcinoma (HCC), or bladder cancer.

[0097] E63. The IL-12 variant, anti-PD1 antibody, fusion protein or method of any one of E59 to E62, wherein the cancer was previously treated with a PD(L)1 inhibitor different from the anti-PD1 antibody of any one of E21 to E35.

[0098] E64. The IL-12 variant, anti-PD1 antibody, fusion protein or method of any one of E59 to E62, wherein the cancer is treated in combination with a PD(L)1 inhibitor that is different from the anti-PD1 antibody of any one of E21 to E35.

[0099] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0100] All references cited herein, including patent applications, patent publications, UniProtKB accession numbers, are hereby incorporated by reference as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0101] The techniques and methods described or referenced herein are generally well understood by those skilled in the art and are routinely applied using conventional methods, such as the widely used methodologies described in Sambrook et al, Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds., (2003)); SERIES METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis(MJGait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:ALaboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture Laboratory Procedures (A.Doyle, JBGriffiths, and DGNewell, eds., 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds); Gene Transfer Vectors for Mammalian Cells (JMMiller and M.P. Calos, eds., 1987); PCR: The PolymeraseChain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (JEColigan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P.Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: APractical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: APractical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow andD.Lane(Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and its updated versions.

[0102] definition

[0103] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by one of ordinary skill in the art.

[0104] As used herein, the singular forms "a", "an" and "the" include plural references unless otherwise specified. For example, "an" antibody includes one or more antibodies.

[0105] When aspects or embodiments of the invention are described in terms of Markush groups or other alternative groupings, the invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, but also the main group lacking one or more of the group members. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

[0106] Any examples following the term "such as" are not meant to be exhaustive or limiting.

[0107] As used herein, the term "about" when used to modify a numerically defined parameter (e.g., the dosage of an IL-12 variant or fusion protein) means that the parameter may be as much as 10% lower or higher than the specified value of the parameter. For example, a dosage of about 5 mg means 5% ± 10%, i.e., it may vary between 4.5 mg and 5.5 mg.

[0108] "Antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a target such as a polypeptide, carbohydrate, polynucleotide, lipid, etc. through at least one antigen binding site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody (e.g., monospecific antibody, bispecific antibody), and includes a portion of a complete antibody that retains the ability to bind to a given antigen (e.g., "antigen-binding fragment"), as well as any other modified configuration of an immunoglobulin molecule that contains an antigen binding site.

[0109] Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular class. Immunoglobulins can be divided into different classes based on the antibody amino acid sequence of the constant region of their heavy chains (HC). There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG 1 IgG 2 IgG 3 IgG 4 , IgA 1 and IgA 2 The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0110] Examples of antibody antigen-binding fragments and modified configurations include: (i) Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments comprising two Fab fragments connected by a disulfide bridge in the hinge region); and (iii) Fv fragments consisting of the VL and VH domains of a single antibody arm. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods to enable them to be made into a single protein chain in which the VL and VH regions are paired to form monovalent molecules (called single-chain Fv (scFv)); see, for example, Bird et al., Science 1988; 242: 423-426 and Huston et al., Proc. Natl. Acad. Sci. 1988 USA 85: 5879-5883. Other forms of single-chain antibodies, such as diabodies, are also contemplated.

[0111] In addition, antibodies lacking a C-terminal lysine (K) amino acid residue on the heavy chain polypeptide are also contemplated (e.g., human IgG1 heavy chains contain a terminal lysine). As is known in the art, the C-terminal lysine is sometimes sheared during antibody production, resulting in antibodies lacking a C-terminal lysine in the heavy chain. Alternatively, nucleic acids can be used to generate antibody heavy chains that do not contain a C-terminal lysine.

[0112] The "variable region" of an antibody refers to the variable region of an antibody light chain alone or in combination or the variable region of an antibody heavy chain. As known in the art, the variable regions of heavy and light chains are each composed of four framework regions (FR) connected by three complementary determining regions (CDR) (also referred to as hypervariable regions), and contribute to the formation of the antigen binding site of an antibody. If a variant of a subject variable region is desired, particularly in an amino acid residue outside the CDR region (i.e., in the framework region), there is a replacement, then suitable amino acid replacement, preferably conservative amino acid replacement can be identified by comparing the subject variable region with the variable region of other antibodies, and the other antibody variable regions contain CDR1 and CDR2 sequences (Chothia and Lesk, J Mol Biol 196 (4): 901-917, 1987) of the same standard category as the subject variable region.

[0113] In certain embodiments, the identification of the residues that clearly describe the CDR and comprise the antibody binding site is completed by resolving the structure of the antibody or resolving the structure of the antibody-ligand complex. In certain embodiments, this can be completed by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, a variety of analytical methods can be used to identify or approximate CDR regions. In certain embodiments, a variety of analytical methods can be used to identify or approximate CDR regions. Examples of this method include but are not limited to Kabat definition, Chothia definition, AbM definition, contact definition, extension definition, and conformational definition.

[0114] The Kabat definition is a standard for numbering residues in antibodies and is often used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. The extended definition is a combination of the Kabat and Chothia definitions. The AbM definition uses an integrated set of computer programs produced by the Oxford Molecular Group to model antibody structures. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM TM,A Computer Program for Modeling Variable Regions of Antibodies,"Oxford,UK; Oxford Molecular,Ltd. AbM definition uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies based on primary sequence, as described, for example, in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Contact definition is based on analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as "conformational definition" of CDRs, the positions of CDRs can be identified as residues that make enthalpic contributions to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166. There are other CDR boundary definitions that may not strictly follow one of the above methods, but will still overlap with at least a portion of the Kabat CDR, although they can be shortened or extended based on predictions or experimental results that a particular residue or residue group will not significantly affect antigen binding. As used herein, CDRs can refer to CDRs defined by any method known in the art (including a combination of methods). The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, CDRs can be defined according to any one or more of the Kabat, Chothia, extension, AbM, contact or conformational definitions.

[0115] The "constant region" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination. The IgG heavy chain constant region contains three consecutive immunoglobulin domains (CH1, CH2, and CH3) with a hinge region between the CH1 and CH2 domains. The IgG light chain constant region contains a single immunoglobulin domain (CL).

[0116] "Fc domain" refers to the portion of an immunoglobulin (Ig) molecule associated with a crystallizable fragment obtained by papain digestion of the Ig molecule. As used herein, the term relates to the 2-chain constant regions of an antibody, each chain excluding the first constant region immunoglobulin domain. There are two "Fc chains" (e.g., "first Fc chain" and "second Fc chain") within the Fc domain. "Fc chain" generally refers to the C-terminal portion of an antibody heavy chain. Thus, the Fc chain refers to the last two constant region immunoglobulin domains (CH2 and CH3) of IgA, IgD, and IgG heavy chains, and the last three constant region immunoglobulin domains of IgE and IgM heavy chains, and optionally the flexible hinge at the N-terminus of these domains.

[0117] Although the boundaries of the Fc chain can vary, a human IgG heavy chain Fc chain is generally defined as comprising residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index of Edelman et al., Proc. Natl. Acad. Sci. USA 1969; 63(1):78-85 and as described by Kabat et al., 1991. Typically, an Fc chain comprises about amino acid residues 236 to about 447 of the constant region of a human IgG1 heavy chain. "Fc chain" can refer to such a polypeptide in isolation or in the context of a larger molecule (e.g., in an antibody heavy chain or Fc fusion protein).

[0118] A "functional" Fc domain refers to an Fc domain that has at least one effector function of a native sequence Fc domain. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors) and B cell activation, etc. Such effector functions generally require an Fc domain in combination with a binding domain (e.g., an antibody variable region) and can be assessed using various assays known in the art for assessing such antibody effector functions.

[0119] A "native sequence" Fc chain refers to an Fc chain comprising an amino acid sequence identical to the amino acid sequence of an Fc chain found in nature. A "variant" Fc chain comprises an amino acid sequence that differs from a native sequence Fc chain by at least one amino acid modification.

[0120] "Monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, compared to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates that the characteristics of the antibody are derived from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by the recombinant DNA method described in, for example, U.S. Patent No. 4,816,567. In another example, monoclonal antibodies can be separated from phage libraries, such as those produced using the technology described in McCafferty et al., 1990, Nature 348:552-554.

[0121] "Human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or an antibody prepared using any technique for preparing a fully human antibody. For example, a fully human antibody can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulins, or by display techniques for libraries (e.g., phage, yeast, or ribosomes) used to prepare fully human antibodies. This definition of a human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues.

[0122] A "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0123] "Humanized" antibodies refer to non-human (e.g., mouse) antibodies that are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the CDRs of the receptor are replaced with residues from the CDRs of non-human species (donor antibodies), such as mice, rats, or rabbits, with the desired specificity, affinity, and capacity. Humanized antibodies may include residues that are not found in the receptor antibody nor in the imported CDR or framework sequences, but these residues are included to further refine and optimize antibody performance.

[0124] "Antigen" refers to a molecular entity used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen or to screen expression libraries (e.g., phage, yeast or ribosome display libraries, etc.) for antibody selection. Herein, the term antigen is broader and is generally intended to include target molecules that are specifically recognized by antibodies, and therefore includes fragments or mimetics of molecules in the immunization process for producing antibodies or in the screening of libraries for selecting antibodies.

[0125] "Epitope" refers to the antigenic site or region to which an antibody specifically binds, such as a site or region comprising residues that interact with the antibody, as determined by any method well known in the art. Many methods are known in the art for mapping and characterizing the position of epitopes on proteins, including crystal structures of antibody-antigen complexes, competitive assays, gene fragment expression assays, epitope mapping, and synthetic peptide-based assays, such as Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999, Chapter 11. In addition or in addition, in the process of revealing, the generation and characterization of antibodies can clarify information about the desired epitope. Based on this information, antibodies that bind to the same epitope can be competitively screened.

[0126] In addition, by using overlapping peptides derived from antigens and determining the combination of antibodies, the epitope of antibody binding can be determined in system screening. According to gene fragment expression assay, the open reading frame encoding the antigen can be randomly or fragmented by a specific genetic construction, and the reactivity of the expressed fragment of the antigen and the antibody to be tested is determined. For example, gene fragments can be produced by PCR, then transcribed and translated into protein in vitro in the presence of radioactive amino acids. The combination of antibodies and radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.

[0127] Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In other examples, antigenic mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues that are required, sufficient, or essential for epitope binding.

[0128] In the most detailed level, the epitope of the interaction between antigen and antibody can be defined by the spatial coordinates of the atomic contacts existing in the antigen-antibody interaction and the information of its relative contribution to the binding thermodynamics. In a less detailed level, the epitope can be characterized by the spatial coordinates of the atomic contacts between the antigen and antibody. In a further less detailed level, the epitope can be characterized by the amino acid residues it comprises, as defined by specific criteria, such as the distance between the atoms (such as heavy atoms, i.e. non-hydrogen atoms) in the antibody and the antigen. In a further less detailed level, the epitope can be characterized by function, such as by combining with competition with other antibodies. The epitope can also be more generally defined as comprising amino acid residues, wherein being replaced by another amino acid will change the interactive features (such as using alanine scanning) between the antibody and the antigen.

[0129] From the fact that descriptions and definitions of epitopes are obtained at different levels of detail, depending on the epitope mapping method used, it can be concluded that comparisons of epitopes of different antibodies on the same antigen can similarly be performed at different levels of detail.

[0130] Epitopes described at the amino acid level, such as those determined by X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (H / D-MS), are said to be identical if they contain the same series of amino acid residues. If the epitopes share at least one amino acid, the epitopes are said to overlap. If the epitopes do not share amino acid residues, the epitopes are said to be independent (unique).

[0131] Another method that can be used to characterize antibodies is to use competition assays with other antibodies known to bind to the same antigen to determine whether the antibody of interest binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art. If the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody excludes the simultaneous or continuous binding of another antibody, then epitopes characterized by competitive binding are considered to be overlapping. If the antigen can accommodate the binding of two corresponding antibodies at the same time, then the epitopes are said to be independent (unique).

[0132] Epitopes can be linear or conformational. In a linear epitope, all interaction points between a protein and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises discontinuous polypeptides (or amino acids) within an antigenic protein to which an antibody specific for the epitope binds.

[0133] The term "binding affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the dissociation constant (K D ). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. In particular, the term "binding affinity" refers to the dissociation rate of a specific antigen-antibody interaction. D is the dissociation rate (also called the “dissociation rate (k off )" or "k d ”) and the binding rate (or “binding rate (k on )" or "k a ”). Therefore, K D Equal to k off / k on (or k d / k a ) and expressed as molar concentration (M). It can be seen that K D The smaller the value, the stronger the binding affinity. D Compared with 1 μM K D Indicates weak binding affinity. D The K value can be determined using methods well established in the art. D An exemplary method of determining the K of an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system such as a BIACORE system. BIACORE kinetic analysis involves analyzing the binding and dissociation of an antigen on a chip having an immobilized molecule (e.g., a molecule comprising an epitope binding domain) on the surface. D Another method is to use bio-layer interferometry, which is usually used Technology (Octet QK e System, ForteBio). Alternatively or additionally, the KinExA (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, ID) may also be used.

[0134] "Monospecific antibody" refers to an antibody that contains one or more antigen binding sites per molecule, such that any and all binding sites of the antibody specifically recognize the same epitope on the antigen. Therefore, where a monospecific antibody has more than one antigen binding site, the binding sites compete with each other for binding to one antigen molecule.

[0135] "Bispecific antibody" refers to a molecule that has binding specificity for at least two different epitopes. In some embodiments, a bispecific antibody can bind to two different antigens simultaneously. In other embodiments, the two different epitopes can reside on the same antigen.

[0136] The term “half maximal effect concentration (EC 50 )” refers to the concentration of therapeutic agent that causes a response intermediate between baseline and maximum after a specified exposure time. The therapeutic agent can cause inhibition or stimulation. EC 50 The value is commonly used and is used herein as a measure of potency.

[0137] "Agonist" refers to a substance that promotes (ie, induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist encompasses substances (eg, antibodies) that bind to a molecule to promote the activity of that molecule.

[0138] "Antagonist" refers to a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effectiveness of another molecule, such as a receptor. The term antagonist encompasses substances (eg, antibodies) that bind to a molecule to prevent or reduce the activity of that molecule.

[0139] The term "competition" as used herein for antibodies refers to the first antibody binding epitope in a sufficiently similar manner to the binding of the second antibody, so that the result of the second antibody binding to its cognate epitope is detectably reduced when the first antibody is present, compared to the binding of the second antibody when the first antibody is not present. Another case is that, in the presence of the second antibody, the binding of the first antibody to its epitope can also be detectably reduced, which can be but not necessarily the case. In other words, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, when each antibody can detectably inhibit the binding of another antibody to its cognate epitope or ligand, whether it is the same, greater or lesser degree, the antibodies are considered to "cross-compete" with each other to bind to their respective epitopes. Competitive antibodies and cross-competitive antibodies are all encompassed by the present invention. Regardless of the mechanism of such competition or cross-competition (e.g., steric hindrance, conformational changes, or binding to a common epitope or part thereof), based on the teachings provided herein, those skilled in the art will understand that such competition or cross-competitive antibodies are also encompassed and can be used in the methods disclosed herein.

[0140] Standard competition assays can be used to determine whether two antibodies compete with each other. A suitable antibody competition assay involves the use of Biacore technology, which can use surface plasmon resonance (SPR) technology, typically using a biosensor system (e.g., BIACORE system) to measure the degree of interaction. For example, SPR can be used for in vitro competitive binding inhibition assays to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay method for measuring antibody competition uses an ELISA-based method.

[0141] "Fc receptor" (FcR) refers to a receptor that binds to the Fc domain of an antibody. In some embodiments, FcR is a natural human FcR. In some embodiments, FcR binds to IgG antibodies (gamma receptors) and includes receptors of FcgRI, FcgRII, and FcgRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcgRII receptors include FcgRIIA ("activation receptor") and FcgRIIB ("inhibition receptor"), which have similar amino acid sequences, the main difference being their cytoplasmic domains. The activation receptor FcgRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcgRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 1997; 15: 203-234). For review of FcRs, see, e.g., Ravetch and Kinet, Annu. Rev. Immunol 1991; 9:457-92; Capel et al., Immunomethods 1994; 4:25-34; and de Haas et al., J. Lab. Clin. Med. 1995; 126:330-41. Other FcRs, including those to be identified in the future, are encompassed by the term "Fc receptor" herein. The term "Fc receptor" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 1976; 117:587 and Kim et al., J. Immunol. 1994; 24:249) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 1997; 18(12):592-598; Ghetie et al., Nature Biotechnology, 1997; 15(7):637-640; Hinton et al., J. Biol. Chem. 2004; 279(8):6213-6216; WO 2004 / 92219).

[0142] "Fragments" or "portions" of antibodies or polypeptides can be prepared by truncation, for example, by removing one or more amino acids from the amino terminus, carboxyl terminus, or both ends of a polypeptide. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 100 or more amino acids can be removed from the amino terminus, carboxyl terminus, or both ends of a polypeptide to produce a fragment or portion. A fragment or portion can be prepared by deleting one or more amino acids from a polypeptide. A fragment or portion can be prepared by deleting one or more amino acids from a polypeptide and removing one or more amino acids from the amino terminus, carboxyl terminus, or both ends of a polypeptide.

[0143] "Effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, effector cells express at least FcgRIII and perform ADCC effector functions. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as from blood.

[0144] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells bearing antigens, which are then killed with cytotoxins. NK cells, the main cells that mediate ADCC, express only FcgRIII, while monocytes express FcgRI, FcgRII, and FcgRIII. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay is performed, such as the method described in U.S. Pat. Nos. 5,500,362, 5,821,337, or 6,737,056. Useful effector cells for such assays include PBMCs and NK cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model, such as disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 1998; 95: 652-656. Additional antibodies with altered Fc domain amino acid sequences and increased or decreased ADCC activity are described, for example, in U.S. Pat. No. 7,923,538 and U.S. Pat. No. 7,994,290.

[0145] The term "altered" FcR binding affinity or ADCC activity refers to an antibody having an enhanced or diminished activity of one or more of FcR binding activity or ADCC activity compared to a parent antibody, wherein the antibody differs from the parent antibody in at least one structural aspect. An antibody that "exhibits increased binding" to an FcR binds to at least one FcR with better affinity than the parent antibody. An antibody that "exhibits decreased binding" to an FcR binds to at least one FcR with less affinity than the parent antibody. Such an antibody that exhibits decreased binding to an FcR may have little or no appreciable binding to an FcR, e.g., 0-20% binding to an FcR compared to a native sequence IgG Fc domain.

[0146] "Host cell" refers to an individual cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0147] "Vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest (e.g., antibody encoding genes) in a host cell. Examples of vectors include, but are not limited to, plasmids and viral vectors, and may include naked nucleic acids, or may include nucleic acids associated with delivery auxiliary materials (e.g., cationic condensing agents, liposomes, etc.). The vector may include DNA or RNA. "Expression vector" as used herein refers to a vector comprising at least one polypeptide encoding gene, at least one regulatory element associated with the transcription or translation of the gene (e.g., a promoter sequence, a poly (A) sequence). Typically, the vector used herein contains at least one antibody encoding gene, and one or more regulatory elements or selection markers. The vector components may include, for example, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (e.g., promoters, enhancers, and terminators). For translation, one or more translation control elements may also be included, such as a ribosome binding site, a translation start site, and a stop codon.

[0148] An "isolated" molecule (e.g., an antibody) is one that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., species, cells from which it is expressed, libraries, etc., (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a chemically synthesized molecule or a molecule expressed in a cellular system different from the system from which it naturally originates would be "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art.

[0149] "Polypeptide" or "protein" (used interchangeably herein) refers to an amino acid chain of any length. The chain may be linear or branched. The chain may contain one or more modified amino acids. The term also encompasses amino acid chains that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that the polypeptide may exist as a single chain or in related chains.

[0150] "Polynucleotide" or "nucleic acid" (used interchangeably herein) refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure can be modified before or after chain assembly. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing side chain moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those with modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl group typically present in the sugar may be replaced by, for example, a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional connections to additional nucleotides, or may be conjugated to a solid support. The 5' and 3' terminal OH may be phosphorylated or partially substituted by an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleosides.

[0151] "Conservative substitution" refers to replacing an amino acid with a biologically, chemically or structurally similar residue. Biological similarity means that the substitution will not destroy biological activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine and serine or similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Specific examples include substitution with another hydrophobic residue, such as isoleucine, valine, leucine or methionine, or substitution with a polar residue, such as arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine, serine for threonine, etc. Specific examples of conservative substitutions include substitution of hydrophobic residues such as isoleucine, valine, leucine or methionine with each other, polar residues for another residue, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, etc. Conservative amino acid substitutions generally include, for example, substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Exemplary potential conservative substitutions include the following amino acid pairs that can be substituted: Ala / Val; Arg / Lys; Asn / Gln; Asp / Glu; Cys / Ser; Gln / Asn; Glu / Asp; Gly / Ala; His / Arg; Ile / Leu; Met / Leu; Phe / Tyr; Pro / Ala; Ser / Thr; Trp / Tyr; Val / Leu.

[0152] The term "identity" or "identical" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (e.g., DNA molecules or RNA molecules) or between polypeptide molecules. "Identity" measures the percentage of identical matches between two or more sequences, gapped alignments solved by a specific mathematical model (e.g., algorithm) of computer programs well known in the art.

[0153] For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by comparing the two sequences for the purpose of optimal comparison (for example, a gap can be introduced in one or both of the first and second sequences for optimal comparison, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When the position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, and considering the number of gaps and the length of each gap, these gaps need to be introduced to achieve the optimal comparison of the two sequences. The comparison of sequences and the determination of the percent identity between the two sequences can be accomplished using a mathematical algorithm.

[0154] To determine percent identity, sequences can be aligned using methods and computer programs available on the National Center for Biotechnology Information (NCBI) World Wide Web, including BLAST. Other alignment programs include Bioinformatics software suite program( Inc., Madison, WI). Another alignment algorithm is FASTA, which can be found in the Genetics Computer Group (GCG) software package (Madison, Wis., USA). Other alignment techniques are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is an algorithm that allows gaps in sequence alignments. See Meth. Mal. Biol. 70: 173-187 (1997). In addition, sequences can be aligned using the GAP program using the Needleman and Wunsch alignment method. See J. Mal. Biol. 48: 443-453 (1970).

[0155] In addition, it is interesting to use the local homology algorithm of Smith and Waterman (1981, Advances in Applied Mathematics 2:482-489) to determine the BestFit program of sequence homogeneity. The gap generation penalty is usually in the range of 1 to 5, usually 2 to 4, and in some embodiments 3. The gap extension penalty is usually in the range of about 0.01 to 0.20, and is 0.10 in some cases. The program has the default parameters determined by the sequence compared by input. Preferably, the default parameters determined using the program are used to determine sequence homogeneity. This program can also be derived from Genetics Computing Group (GCG) software package (Madison, WI, USA).

[0156] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. FastDB calculates percent sequence identity according to the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; merging penalty: 30.0.

[0157] The terms "increase," "improve," "decrease," or "reduction" refer to values ​​relative to a baseline measurement, such as a measurement of the same individual before the start of a treatment as described herein, or a measurement of a control individual or subject (or multiple control individuals or subjects) in the absence of a treatment as described herein. In some embodiments, a "control individual" is an individual with the same form of disease or injury as the individual being treated. In some embodiments, a "control individual" is an individual who does not have the same form of disease or injury as the individual being treated.

[0158] The term "excipient" refers to any material that is combined with an active ingredient of interest (e.g., an antibody) to retain biological activity in the active ingredient. The selection of an excipient depends to a large extent on a variety of factors, such as the mode of administration, the effect of the excipient on solubility and stability, and the properties of the dosage form. As used herein, "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, vehicles, diluents, etc. that are physiologically compatible. Examples of excipients include one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof, and isotonic agents such as sugars, sodium chloride, or polyols such as mannitol or sorbitol may be included in the composition.

[0159] The term "treat" refers to any type of treatment, such as alleviating, relieving or slowing the progression of a patient's disease, disorder or condition or any tissue damage associated with the disease. In some embodiments, the disease, disorder or condition is cancer.

[0160] The term "prevention" refers to preventing a disease, condition, or disorder in an individual who may be susceptible to the disease, condition, or disorder but has not yet experienced or exhibited the pathology or symptomatology of the disease. In some embodiments, prevention is assessed on a population basis, whereby an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the development, frequency, or intensity of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. The prevention may be considered complete when the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

[0161] The terms "subject," "individual," or "patient" (used interchangeably herein) refer to any animal, including mammals. Mammals according to the present invention include canines, felines, bovines, caprines, equines, ovines, porcines, rodents, lagomorphs, primates, humans, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects can be of any gender and at any stage of development. In some embodiments, the subject is a patient suffering from cancer.

[0162] The term "therapeutically effective amount" refers to the amount of an active ingredient that will elicit the biological or medical response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which biological or medical response may include one or more of the following:

[0163] (1) preventing a disease; e.g., preventing a disease, disorder, or condition in an individual who may be susceptible to the disease, disorder, or condition but who does not yet experience or exhibit the pathology or symptomology of the disease;

[0164] (2) inhibiting a disease; e.g., inhibiting the disease, disorder, or condition in an individual who is experiencing or exhibiting the pathology or symptomology of the disease, disorder, or condition (i.e., preventing or slowing the further development of the pathology or symptomology); and

[0165] (3) ameliorating a disease; e.g., ameliorating the disease, disorder, or condition in an individual who is experiencing or exhibiting the pathology or symptomology of the disease, disorder, or condition (i.e., reversing the pathology or symptomology).

[0166] IL-12 variants

[0167] In some embodiments, provided herein are interleukin 12 (IL-12) variants. IL-12 variants are also referred to as IL-12 "mutants."

[0168] IL-12 is a heterodimer containing two subunits: p35 (also known as IL-12α; encoded by the IL-12A gene) and p40 (also known as IL-12β; encoded by the IL-12B gene). These two IL-12 subunits can form an intersubunit disulfide bond between C177 of p40 and C74 of p35.

[0169] The amino acid sequence of the mature wild-type human IL-12 p35 subunit is provided herein as SEQ ID NO: 1: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 1).

[0170] The mature human p35 subunit (SEQ ID NO: 1) is produced by the full-length p35 polypeptide, which also includes a 22-amino acid signal peptide that is cleaved during the intracellular processing of the initial translated precursor protein. The full-length human p35 amino acid sequence including the signal peptide can be obtained as UniProt accession number P29459, and is provided herein as SEQ ID NO: 28 (the signal peptide is underlined):

[0171] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:28)

[0172] All references herein to specific amino acid numbers in the IL-12p35 amino acid sequence refer to the amino acid position in the mature IL-12p35 sequence lacking the signal peptide (not to the amino acid position in the precursor full-length protein). For example, the amino acid "R1" in the IL-12p35 amino acid sequence refers to the arginine (R) at the first position in SEQ ID NO:1.

[0173] The amino acid sequence of the mature wild-type human IL-12p40 subunit is provided herein as SEQ ID NO: 2: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 2).

[0174] The mature human p40 subunit (SEQ ID NO: 2) is produced by the full-length p40 polypeptide, which also includes a 22-amino acid signal peptide that is cleaved during the intracellular processing of the initial translated precursor protein. The full-length human p40 amino acid sequence including the signal peptide can be obtained as UniProt accession number P29460, and is provided herein as SEQ ID NO: 29 (the signal peptide is underlined):

[0175] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSA ERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:29)

[0176] All references herein to specific amino acid numbers in the IL-12p40 amino acid sequence refer to the amino acid position in the mature IL-12p40 sequence lacking the signal peptide (not to the amino acid position in the precursor full-length protein). For example, amino acid "W2" in the IL-12p40 amino acid sequence refers to the tryptophan (W) at the second position in SEQ ID NO:2.

[0177] As used herein, IL-12 "variants" or "mutants" refer to any IL-12 molecule containing at least one amino acid change in at least one of the p35 or p40 subunits compared to the amino acid sequence of the wild-type mature p35 subunit (SEQ ID NO: 1) or the wild-type mature p40 subunit (SEQ ID NO: 2). In some embodiments, the IL-12 variants provided herein may have at least one amino acid change in both the p35 and p40 subunits compared to the amino acid sequence of the wild-type mature p35 (SEQ ID NO: 1) or p40 (SEQ ID NO: 2).

[0178] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution at one or more of the following positions in SEQ ID NO: 1 (p35 subunit): F39, I52, or Y167. In some embodiments, the F39 substitution is F39R or F39A. In some embodiments, the I52 substitution is I52E, I52R, or I52H. In some embodiments, the Y167 substitution is Y167A.

[0179] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution at one or more of the following positions in SEQ ID NO: 2 (p40 subunit): K85 or D93. In some embodiments, the K85 substitution is K85E. In some embodiments, the D93 substitution is D93L.

[0180] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution at one or more of the following positions in SEQ ID NO: 1 (p35 subunit): F39, I52, or Y167, and an amino acid substitution at one or more of the following positions in SEQ ID NO: 2 (p40 subunit): K85 or D93. In some embodiments, the F39 substitution is F39R or F39A. In some embodiments, the I52 substitution is I52E, I52R, or I52H. In some embodiments, the Y167 substitution is Y167A. In some embodiments, the K85 substitution is K85E. In some embodiments, the D93 substitution is D93L.

[0181] In some embodiments, provided herein are IL-12 variants with reduced activity, wherein the IL-12 variant is a variant designated as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, H25, H30, H31 or H32 in Table 10 of Example 1, and wherein the corresponding variant has a corresponding mutation in one or both of the p35 and p40 subunits as shown in Table 10.

[0182] In some embodiments, the IL-12 variants provided herein have reduced activity. As used herein, the "reduced activity" of IL-12 variants refers to the activity reduced compared to the activity of the corresponding wild-type IL-12 (e.g., wild-type human IL-12). The "activity" of IL-12 can be evaluated by any suitable assay known in the art for measuring IL-12 activity. For example, IL-12 activity can be evaluated by measuring STAT4 phosphorylation (pSTAT4) in response to IL-12 exposure in cells. STAT4 phosphorylation is an early downstream effect of receptor dimerization induced by IL-12, so it can be used as a receptor proximal reading of IL-12 activity. In another example, IL-12 activity can be evaluated by checking type 1 T helper cells ("Th1") related gene transcription (e.g., IFNγ gene transcription). pSTAT4 causes Th1 related gene transcription to be upregulated, so IFNγ (or other Th1 related genes) can be used as a downstream reading of IL-12 activity. In another example, IL-12 activity can be assessed indirectly by measuring the affinity of the IL-12 variant for the IL-12 receptor.

[0183] IL-12 variants with reduced activity may also be described as "reduced potency" IL-12 variants, "partial agonists," and the like.

[0184] In some embodiments, the IL-12 variants provided herein have reduced binding to the IL-12 receptor compared to the binding of wild-type IL-12 to the IL-12 receptor. The IL-12 receptor is a heterodimer containing subunits IL-12Rbeta1 (for details on human IL-12Rbeta1, see UniProt ID NO: P42701) and IL-12Rbeta2 (for details on human IL-12Rbeta2, see UniProt ID NO: Q99665). IL-12 variants with reduced binding to the IL-12 receptor can be useful, for example, in cases where the IL-12 variant still binds to the IL-12 receptor with sufficient affinity to activate the receptor in certain circumstances, but it is less activated than wild-type IL-12 to the IL-12 receptor. Although IL-12 activity may be therapeutically useful to activate the subject's immune system, excessive IL-12 activity may be harmful to the patient when the immune response is overstimulated, which may lead to treatment-related adverse events (TRAEs), such as cytokine release syndrome (CRS).

[0185] In some embodiments, the activity of the IL-12 variants provided herein is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the activity of the same amount of the corresponding wild-type IL-12 molecule when tested under the same experimental conditions.

[0186] In some embodiments, the activity of the IL-12 variants provided herein is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, 99.99% or more, compared to the activity of the same amount of the corresponding wild-type IL-12 molecule when tested under the same experimental conditions.

[0187] In some embodiments, the activity of the IL-12 variants provided herein is reduced by 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 50-fold or more, 100-fold or more, 500-fold or more, 1000-fold or more, 5000-fold or more, 10000-fold or more, 15000-fold or more, 20000-fold or more, 23000-fold or more, 25000-fold or more, 50000-fold or more, or 100,000-fold or more compared to the same amount of the corresponding wild-type IL-12 molecule when tested under the same experimental conditions.

[0188] In some embodiments, the IL-12 variants provided herein have an affinity for the IL-12 receptor that is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the affinity of the corresponding wild-type IL-12 molecule for the IL-12 receptor when tested under the same experimental conditions.

[0189] In some embodiments, the IL-12 variants provided herein have an affinity for the IL-12 receptor that is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, 99.99% or more compared to the affinity of the corresponding wild-type IL-12 molecule for the IL-12 receptor when tested under the same experimental conditions.

[0190] In some embodiments, the IL-12 variants provided herein have an affinity for the IL-12 receptor that is reduced by 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 50-fold or more, 100-fold or more, 500-fold or more, 1000-fold or more, 5000-fold or more, 10000-fold or more, 15000-fold or more, 20000-fold or more, 23000-fold or more, 25000-fold or more, 50000-fold or more, or 100,000-fold or more compared to the affinity of the corresponding wild-type IL-12 molecule for the IL-12 receptor when tested under the same experimental conditions.

[0191] Exemplary IL-12 variants provided herein include those shown in Example 1 and described in the claims and enumerated embodiments. IL-12 variants include, for example, an H10 mutein comprising the p35 amino acid sequence of SEQ ID NO: 3 and the p40 amino acid sequence of SEQ ID NO: 4, as shown in Table 1.

[0192] Table 1

[0193]

[0194]

[0195] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution at one or more of the following positions in SEQ ID NO: 1 (p35 subunit): F39, I52 or Y167, wherein the variant comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater sequence identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the F39 substitution is F39R or F39A. In some embodiments, the I52 substitution is I52E, I52R or I52H. In some embodiments, the Y167 substitution is Y167A.

[0196] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution at one or more of the following positions in SEQ ID NO: 2 (p40 subunit): K85 or D93, wherein the variant comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the K85 substitution is K85E. In some embodiments, the D93 substitution is D93L.

[0197] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution at one or more of the following positions in SEQ ID NO: 1 (p35 subunit): F39, I52, or Y167, and an amino acid substitution at one or more of the following positions in SEQ ID NO: 2 (p40 subunit): K85 or D93, wherein the variant comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the F39 substitution is F39R or F39A. In some embodiments, the I52 substitution is I52E, I52R, or I52H. In some embodiments, the Y167 substitution is Y167A. In some embodiments, the K85 substitution is K85E. In some embodiments, the D93 substitution is D93L.

[0198] In some embodiments, provided herein are IL-12 variants, wherein the variant comprises an amino acid substitution Y167A in SEQ ID NO: 1 (p35 subunit) and an amino acid substitution D93L in SEQ ID NO: 2 (p40 subunit), wherein the variant comprises an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0199] In some embodiments, provided herein are IL-12 variants provided herein linked to another protein, such as an antibody. These molecules are referred to as "IL-12 variant fusion proteins" and will be described in detail later herein.

[0200] Antibodies to PD1

[0201] The present disclosure also provides antibodies that bind to human PD1. PD1 (programmed cell death protein 1; also known as PD-1 and CD279) is an immune checkpoint protein. PD1 is a type 1 transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD1 is expressed on a variety of immune cells such as T cells, B cells, and macrophages. The ligands of PD1 are B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC). PD1 downregulates immune cell activity; therefore, inhibition of PD1 results in increased immune cell activity, such as increased T cell proliferation and activation, increased secretion of IFN, IL-2, and TNF by immune cells, and increased anti-tumor responses of immune cells.

[0202] As used herein, the term "PD1" includes variants, isoforms, homologs, orthologs and paralogs of PD1. In some embodiments, the antibodies disclosed herein cross-react with PD1 from species other than humans, such as PD1 of cynomolgus monkeys, and different forms of PD1. In some embodiments, the antibody may be completely specific to human PD1 and may not exhibit species cross-reactivity (e.g., not binding to mouse PD1) or other types of cross-reactivity (e.g., not binding to other receptors in the tumor necrosis factor receptor family). As used herein, the term PD1 refers to naturally occurring human PD1, unless the context indicates otherwise. Therefore, "PD1 antibody", "anti-PD1 antibody" or other similar names refer to any antibody (as defined herein) that binds or reacts to PD1, its isoform, fragment or derivative.

[0203] A variety of different anti-PD1 antibodies have been developed for the treatment of cancer, such as nivolumab and pembrolizumab. Typically, these antibodies provide therapeutic effects by reducing the biological activity of PD1, for example, by inhibiting the binding of PD1 to its ligands PDL1 and PDL2. Anti-PD1 antibodies that inhibit the binding of PD1 to one or both of PDL1 and PDL2 are referred to herein as "blocking," "inhibitory," or "antagonistic" anti-PD1 antibodies.

[0204] In contrast, in some embodiments, antibodies are provided herein that bind to PD1 but do not inhibit (or do not completely inhibit) the binding of PDL1 and PDL2 to PD1. These antibodies can bind to PD1 while PD1 binds to PDL1 or PDL2. These antibodies are referred to herein as "non-blocking" anti-PD1 antibodies. Non-blocking anti-PD1 antibodies are useful because of their ability to bind to PD1 (even if they do not inhibit PD1 activity mediated by PD1-PDL1 / PDL2 interactions). For example, non-blocking anti-PD1 antibodies can be used to target molecules connected to non-blocking anti-PD1 antibodies to PD1-expressing cells, such as T cells.

[0205] In some embodiments, a non-blocking anti-PD1 antibody provided herein can bind to PD1 at the same time that PD1 is bound by a blocking anti-PD1 antibody.

[0206] As used herein, the term "PD1 binding" antibody refers to both blocking and non-blocking anti-PD1 antibodies.

[0207] In some embodiments, the anti-PD1 antibodies of the present disclosure include one or both of the following antibodies: i) compete for binding to human PD1 or ii) bind to the same epitope with an antibody comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-PD1 antibodies of the present disclosure include one or both of the following antibodies: i) compete for binding to human PD1 or ii) bind to the same epitope with an antibody comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 17 and the light chain variable region amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-PD1 antibodies of the present disclosure include one or both of the following antibodies: i) compete for binding to human PD1 or ii) bind to the same epitope with an antibody comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 33 and the light chain variable region amino acid sequence of SEQ ID NO: 34.

[0208] The anti-PD-1 antibodies disclosed herein may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab') 2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chains (ScFv), mutants thereof, fusion proteins comprising antibody fragments (e.g., domain antibodies), humanized antibodies, and immunoglobulin molecules of any other modified configurations comprising antigen-binding sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the anti-PD1 antibody is a monoclonal antibody. In some embodiments, the anti-PD1 antibody is a human antibody or a humanized antibody. In some embodiments, the anti-PD1 antibody provided herein is a chimeric antibody. In some embodiments, the anti-PD1 antibody provided herein is of the IgG1 subclass. In some embodiments, the anti-PD1 antibody provided herein has a knob-and-hole mutation in the Fc chain to promote heterodimerization between chains. In some embodiments, the anti-PD1 antibody provided herein has a mutation in the Fc chain to reduce binding affinity to human Fcγ receptors.

[0209] In some embodiments, the present invention provides an antibody or variant thereof having a light chain variable region (VL) sequence and a heavy chain variable region (VH) sequence as shown in Table 2. In Table 2, the underlined sequences are CDR sequences (Kabat definition) and the bold sequences are CDR sequences (Chothia definition).

[0210] Table 2

[0211]

[0212] In some embodiments, provided herein are antibodies comprising a VH as set forth in SEQ ID NO: 7 and a VL as set forth in SEQ ID NO: 8. In some embodiments, provided herein are antibodies comprising a VH as set forth in SEQ ID NO: 17 and a VL as set forth in SEQ ID NO: 18. In some embodiments, provided herein are antibodies comprising a VH as set forth in SEQ ID NO: 33 and a VL as set forth in SEQ ID NO: 34.

[0213] The present invention also provides a CDR portion of a PD1 antibody. The determination of CDR regions is well known to those skilled in the art. It should be understood that in some embodiments, the CDR may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In another method referred to herein as "conformational definition" of CDRs, the positions of CDRs can be identified as residues that contribute enthalpically to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166. In general, "conformational CDRs" include residue positions in Kabat CDRs and Vernier regions that are restricted to maintain the correct loop structure for antibody binding to a specific antigen. The determination of conformational CDRs is well known to those skilled in the art. In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In other embodiments, the CDR is an extended, AbM, conformational, or contact CDR. In other words, in embodiments having more than one CDR, the CDRs can be any of the Kabat, Chothia, extended, AbM, conformational, contact CDRs, or a combination thereof.

[0214] In some embodiments, the antibody comprises three CDRs of the heavy chain variable region shown in Table 2. In some embodiments, the antibody comprises three CDRs of the light chain variable region shown in Table 2. In some embodiments, the antibody comprises three CDRs of the heavy chain variable region shown in Table 2 and three CDRs of the light chain variable region shown in Table 2.

[0215] Table 3 provides examples of CDR sequences for anti-PD1 antibodies provided herein. CDRs not annotated with any particular CDR definition in Table 3 have the same CDR definition according to Chothia, Kabat, and the extended definition.

[0216] Table 3: Anti-PD1 antibodies (mAbs) and their antigen-binding CDR sequences

[0217]

[0218]

[0219] In some embodiments, the anti-PD1 antibodies provided herein comprise three light chain CDRs and three heavy chain CDRs from an antibody as shown in Table 3.

[0220] In some embodiments, the anti-PD1 antibody comprises one or both of i) a full-length heavy chain (with or without a C-terminal lysine) or ii) a full-length light chain. The amino acid sequences of the full-length heavy and light chains of exemplary anti-PD1 antibodies provided herein are shown in Table 4 below.

[0221] Table 4: Heavy and light chain sequences of anti-PD1 mAbs

[0222]

[0223]

[0224]

[0225] In Table 5, the amino acid sequence of "TPP-77658 heavy chain" (SEQ ID NO:5) includes the following annotated features in Fc: effector null mutations L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). The amino acid sequence of "TPP-77658 heavy chain without hole mutation" (SEQ ID NO:51) is the same as SEQ ID NO:5, except that it does not contain the hole-forming mutation; it still contains the effector null mutations L234A, L235A and G237A (underlined). The amino acid sequence of "TPP-77658 heavy chain without hole mutation or effector null mutation" (SEQ ID NO:52) is the same as SEQ ID NO:5, except that it does not contain the hole mutation or effector null mutation of SEQ ID NO:5; instead, it contains the corresponding wild-type amino acids.

[0226] In Table 5, the amino acid sequence of "TPP-76868 heavy chain" (SEQ ID NO: 15) includes the following annotated features in Fc: effector null mutations L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). The amino acid sequence of "TPP-76868 heavy chain without hole mutation" (SEQ ID NO: 53) is the same as SEQ ID NO: 15, except that it does not contain the hole-forming mutations; it still contains the effector null mutations L234A, L235A and G237A (underlined). The amino acid sequence of "TPP-76868 heavy chain without hole mutation or effector null mutation" (SEQ ID NO:54) is identical to SEQ ID NO:15, except that it does not comprise the hole mutation or effector null mutation of SEQ ID NO:15; instead, it contains the corresponding wild-type amino acid.

[0227] In Table 5, the amino acid sequence of "TPP-68807 heavy chain" (SEQ ID NO: 42) includes the following annotated features in Fc: effector null mutations L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). The amino acid sequence of "TPP-68807 heavy chain without hole mutation" (SEQ ID NO: 55) is the same as SEQ ID NO: 42, except that it does not contain the hole-forming mutations; it still contains the effector null mutations L234A, L235A and G237A (underlined). The amino acid sequence of "TPP-68807 heavy chain without hole mutation or effector null mutation" (SEQ ID NO:56) is identical to SEQ ID NO:5, except that it does not contain the hole mutation or effector null mutation of SEQ ID NO:42; instead, it contains the corresponding wild-type amino acid.

[0228] In certain embodiments, the antibodies described herein comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g. IgG 1 IgG 2 IgG 3 or IgG 4 ). In some embodiments, the anti-PD1 antibody provided herein is an IgG1 antibody.

[0229] The present invention encompasses modifications to the variable regions shown in Table 2, the CDRs shown in Table 3, and the heavy and light chain sequences shown in Table 4. For example, the present invention includes antibodies comprising functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with enhanced or reduced activity or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with the desired binding affinity for PD1. Modification of polypeptides is routine practice in the art and does not need to be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, polypeptides with one or more amino acid deletions or additions that do not significantly and adversely change the functional activity, or mature (enhance) the affinity of the polypeptide for its ligand, or use chemical analogs.

[0230] Modification or mutation can also be carried out in framework region or constant region to increase the half-life of antibody provided herein.See, for example, PCT Publication No. WO 00 / 09560. Mutation can also be carried out in framework region or constant region to change the immunogenicity of antibody, to provide a site for covalent or non-covalent binding with another molecule, or to change properties such as complement fixation, FcR combination and antibody-dependent cell-mediated cytotoxicity. In some embodiments, no more than 1 to 5 conservative amino acid substitutions are carried out in framework region or constant region. In other embodiments, no more than 1 to 3 conservative amino acid substitutions are carried out in framework region or constant region. According to the present invention, single antibody can have mutation in any one or more CDR or framework region of variable domains or in constant region.

[0231] In some embodiments, the antibody comprises a modified constant region, which has an increased or decreased binding affinity for human Fcγ receptors, is immunologically inert or partially inert, such as not triggering complement-mediated lysis, not stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), or not activating microglia; or has reduced activity (compared to unmodified antibodies) in any one or more of the following aspects: triggering complement-mediated lysis, stimulating ADCC or activating microglia. Different modifications of the constant region can be used to achieve the optimal level or combination of effector functions. See, e.g., Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624, PCT Publication No. WO99 / 058572.

[0232] For example, in some embodiments, the constant region of the antibodies provided herein is modified to have reduced binding affinity to human Fcγ receptors. These antibodies are also referred to as "effector null" or having "inert Fc domains". Such antibodies may have, for example, one or more of the mutations L234A, L235A, and G237A in the IgG1 CH2 domain to reduce or eliminate effector function (numbered according to EU nomenclature).

[0233] Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation of different sugars, acetylation and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65: 111-128; Wright and Morrison, 1997, TibTECH 15: 26-32). The oligosaccharide side chains of immunoglobulins affect the function of proteins (Boyd et al., 1996, Mol. Immunol. 32: 1311-1318; Wittwe and Howard, 1990, Biochem. 29: 4175-4180) and the intramolecular interactions between the parts of the glycoprotein can affect the conformation and the three-dimensional surface presented by the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on specific recognition structures. It is reported that glycosylation of antibodies can affect antibody-dependent cellular cytotoxicity (ADCC). In particular, antibodies produced by CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing the bisecting GlcNAc formation, were reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

[0234] In some embodiments, the disclosure provides anti-PD1 antibodies containing changes in the variable regions set forth in Table 2, the CDRs set forth in Table 3, or the heavy and light chain sequences set forth in Table 4, wherein such variant polypeptides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of the amino acid sequences disclosed in Tables 2, 3, or 4. These amounts are not meant to be limiting, and increments between the recited percentages are specifically contemplated as part of the disclosure.

[0235] In some embodiments, provided herein are anti-PD1 antibodies comprising VH and VL, wherein antibody VH has an amino acid sequence encoded by a nucleic acid sequence within the nucleic acid sequence of an insert fragment of a plasmid deposited with ATCC having ATCC Accession No. PTA-127517, and antibody VL has an amino acid sequence encoded by a nucleic acid sequence within the nucleic acid sequence of an insert fragment of a plasmid deposited with ATCC having ATCC Accession No. PTA-127519.

[0236] The present invention also encompasses fusion proteins comprising one or more components of the antibodies disclosed herein. In some embodiments, a fusion protein comprising all or part of an anti-PD1 antibody of the present invention connected to another polypeptide can be prepared. In another embodiment, only the variable domain of the anti-PD1 antibody is connected to the polypeptide. In another embodiment, the VH domain of the anti-PD1 antibody is connected to the first polypeptide, and the VL domain of the anti-PD1 antibody is connected to the second polypeptide, which is associated with the first polypeptide in a manner that enables the VH and VL domains to interact to form an antigen binding site. In another embodiment, the VH domain is separated from the VL domain by a linker so that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then connected to the polypeptide of interest. In addition, fusion antibodies can be produced in which two (or more) single-chain antibodies are connected to each other. This is very useful if you want to produce a bivalent or multivalent antibody on a single polypeptide chain, or if you want to produce a bispecific antibody.

[0237] IL-12 variant fusion protein

[0238] In some embodiments, provided herein are IL-12 variants provided herein linked to another protein. These molecules are referred to herein as "IL-12 variant fusion proteins".

[0239] In some embodiments, the IL-12 variant can be linked to various other types of proteins, such as an antibody, another cytokine, an enzyme, or an antibody Fc domain.

[0240] The IL-12 variants provided herein can be connected to another protein in any suitable manner. For example, the IL-12 variant can be directly covalently connected to another protein (e.g., such that the amino acids of IL-12 are directly covalently connected to the amino acids of another protein) or connected via a polypeptide linker. In addition, IL-12 can be connected in any direction relative to other proteins, for example, it can be connected to the N or C terminus of another protein; similarly, the N or C terminus of the IL-12 subunit can be connected to other proteins.

[0241] When IL-12 is connected to another protein, one or two IL-12 subunits may be connected to another protein. For example, if the IL-12 variants provided herein are connected to an antibody, then in some embodiments, one subunit of IL-12 (e.g., p35) may be connected to the first chain of the antibody, and another subunit of IL-12 (e.g., p40) may be connected to the second chain of the antibody. In other embodiments, only one IL-12 subunit (p35 or p40) is connected to the chain of the antibody, and the other unconnected IL-12 subunit is associated in the complex through its interaction with the connected IL-12 subunit.

[0242] In some embodiments, when IL-12 is linked to another protein, IL-12 can be prepared as a single polypeptide chain containing both the p35 and p40 subunits of IL-12. To facilitate expression of the recombinant IL-12 variants provided herein, it may be desirable to combine the sequences of the p35 and p40 subunits into a single polypeptide that can be assembled into a complete IL-12 molecule.

[0243] In some embodiments, the IL-12 variant is linked to an antibody. Linking of the IL-12 variant to the antibody can be used for one or more purposes, such as 1) targeting IL-12 to the location of the antibody target (e.g., if the antibody binds to a cell surface receptor, linking IL-12 to the antibody can target IL-12 to cells with the cell surface receptor) and 2) if the antibody has a therapeutic effect, the IL-12 activity can be combined with the therapeutic effect of the antibody.

[0244] In some embodiments, provided herein is a fusion protein connecting an IL-12 variant provided herein and an anti-PD1 antibody (referred to herein as "IL-12 variant / anti-PD1 fusion protein", "IL-12 variant / anti-PD1 molecule", etc.). In some embodiments, the anti-PD1 antibody is an anti-PD1 antibody provided herein.

[0245] High expression of PD1 is mainly found in CD8-positive and CD4-positive tumor-infiltrating lymphocytes (TILs) and is enriched in the tumor microenvironment (TME) compared with circulating T cell subsets. This anatomical localization and cellular expression profile suggest that targeting IL-12 activity to PD1-positive (PD1+) cells could reduce systemic activity while still achieving strong antitumor immunity. Overall, it is hypothesized that directing IL-12 activity from the periphery to the tumor microenvironment will enhance the antitumor efficacy of IL-12 in PD(L)1 antagonist-naïve and PD(L)1 antagonist-resistant tumors while reducing systemic toxicity.

[0246] PD1 positive cells (such as CD8 positive T cells and CD4 positive T cells) also generally contain IL-12 receptors. Therefore, the combination of the antibody portion of IL-12 variant / anti-PD1 fusion protein with PD1 on immune cells containing IL-12 receptors brings the IL-12 variant in the fusion protein very close to the IL-12 receptor on PD1 positive cells. In other words, the IL-12 variant / anti-PD1 fusion provided herein can bind to both 1) PD1 and 2) IL-12 receptors on the same cell (such as T cells). This is also referred to as "cis targeting" of IL-12 (i.e., targeting IL-12 variants to the same cell bound by the antibody connected to the IL-12 variant). IL-12 variant cis targeting PD1 positive cells has a variety of potential benefits. First, in view of the low affinity of IL-12 variants for IL-12 receptors, IL-12 variants (such as IL-12 variants provided herein) with reduced affinity for IL-12 receptors have low activity for cells that are positive for IL-12 receptors but negative for PD1. IL-12 receptor-positive but PD1-negative (or low PD1) cells are the most common circulating cells / peripheral cells (ie, outside the tumor microenvironment). Therefore, fusion proteins containing IL-12 variants and anti-PD1 antibodies with reduced affinity for IL-12 receptors have minimal activity and associated potential toxicity to peripheral cells that do not express PD1. Secondly, in view of the binding of IL-12 variants / anti-PD1 fusion molecules to PD1, IL-12 variants (such as IL-12 variants provided herein) with reduced affinity for IL-12 receptors can still have effective activity on IL-12 receptor-positive and PD1-positive cells. In this case, the anti-PD1 antibody bound to PD1 effectively maintains the IL-12 variant in close proximity to the IL-12 receptor, such that the IL-12 variant and the IL-12 receptor still interact strongly enough to produce the downstream effects of IL-12 binding to the IL-12 receptor (e.g., increasing CD8 T cell cytotoxicity, promoting CD4 Th1 cell differentiation, inhibiting regulatory T cell (Treg) function, and increasing additional cytokine and chemokine expression, thereby producing various anti-tumor effects, such as recruiting immune cells, inhibiting angiogenesis, and inhibiting tumor growth). It can therefore be said that linking the anti-PD1 antibody to an IL-12 variant with reduced affinity for the IL-12 receptor "rescues" the activity of the IL-12 variant, because the IL-12 variant has low or no activity on the IL-12 receptor unless the IL-12 variant is maintained in close physical proximity to the IL-12 receptor by being linked to the anti-PD1 antibody, which binds to the PD1 molecule on the surface of a cell near the IL-12 receptor on the same cell surface.

[0247] In some embodiments, the IL-12 variant / anti-PD1 fusion protein is selected after optimizing the anti-PD1 binding of the antibody portion of the fusion protein and the IL-12 activity of the IL-12 variant portion of the fusion protein to achieve a selected balance of potency and efficacy of the fusion protein. In some embodiments, the IL-12 variant / anti-PD1 fusion protein provided herein is designed to have one, two, or all three of the following features: 1) PD1-mediated, affinity-driven IL-12 receptor stimulation is preferentially delivered to PD1-positive cells; 2) Compared with the full agonist IL-12 molecule (e.g., wild-type IL-12-Fc fusion molecule), an improved therapeutic index and 3) binding to an epitope on PD1, allowing a PD1 antagonist (e.g., an antibody that blocks the interaction of PD1 with PDL1) to bind to PD1 simultaneously, so that when the IL-12 variant / anti-PD1 fusion protein binds to PD1, the PD1 antagonist activity is maintained.

[0248] Exemplary IL-12 variant / anti-PD1 fusion proteins include those shown in the Examples herein and described in the claims and embodiments.

[0249] In one embodiment, the present invention provides an IL-12 variant / anti-PD1 fusion protein comprising the following features. The anti-PD1 portion of the fusion protein is an anti-PD1 antibody containing two heavy chains and two light chains. One of the heavy chains of the anti-PD1 antibody has a linker sequence at the C-terminus of the chain, which is connected to the N-terminus of the IL-12 variant p35 amino acid sequence so that it forms a single continuous polypeptide, comprising the following components (in order from N-terminus to C-terminus): anti-PD1 heavy chain-linker sequence-IL-12 variant p35 sequence. There is a "knob" mutation in the Fc of one heavy chain, and a "hole" mutation in the Fc of the other heavy chain to promote heterodimerization of the two heavy chains. The p40 subunit of the IL-12 variant is connected to the p35 subunit via a disulfide bond.

[0250] In one embodiment, the IL-12 variant / anti-PD1 fusion protein provided herein is a fusion protein containing an IL-12H10 mutant protein and an anti-PD1 antibody TPP-77658. This fusion protein is also referred to herein as "H10658 fusion". There are a total of 5 separate polypeptides in the H10658 fusion: 1) antibody heavy chain (not linked to the IL-12 peptide); 2) antibody heavy chain linked to p35 of the H10 IL-12 mutant protein; 3) antibody light chain (copy 1); 4) antibody light chain (copy 2); 5) p40 of the IL10 IL-12 mutant protein. Among these 5 separate polypeptides, there are 4 different polypeptide sequences (there are two copies of the antibody light chain in the fusion protein; these two light chains have the same amino acid sequence). The amino acid sequence of the polypeptide in the H10658 fusion is shown in Table 5 below.

[0251] Table 5

[0252]

[0253] The amino acid sequence of the H10658 fusion shown in Table 5 includes the following annotated features. In the heavy chain TPP-77658 sequence (SEQ ID NO: 5), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). In the heavy chain TPP-77658 of p35 fused to the H10 sequence (SEQ ID NO: 25), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), mutations forming the "knob" of the knob-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence of p35 connecting the heavy chain and H10 [SGGGGS GGGGSGGGG (SEQ ID NO: 27)]. The C-terminal lysine of SEQ ID NO: 5 is optional.

[0254] In one embodiment, the IL-12 variant / anti-PD1 fusion protein provided herein is a fusion protein containing an IL-12H10 mutant protein and an anti-PD1 antibody TPP-76868. This fusion protein is also referred to herein as an "H10868 fusion". There are a total of 5 separate polypeptides in the H10868 fusion: 1) antibody heavy chain (not linked to an IL-12 polypeptide); 2) antibody heavy chain linked to p35 of an H10 IL-12 mutant protein; 3) antibody light chain (copy 1); 4) antibody light chain (copy 2); 5) p40 of an IL10 IL-12 mutant protein. Among the 5 separate polypeptides, there are 4 different polypeptide sequences (there are two copies of the antibody light chain in the fusion protein; the two light chains have the same amino acid sequence). The amino acid sequences of the polypeptides in the H10868 fusion are shown in Table 6 below.

[0255] Table 6

[0256]

[0257] The amino acid sequence of the H10868 fusion shown in Table 6 includes the following annotated features. In the heavy chain TPP-76868 sequence (SEQ ID NO: 15), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). In the heavy chain TPP-76868 fused to p35 of H10 sequence (SEQ ID NO: 26), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), mutations forming the "knob" of the knob-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence connecting the heavy chain and p35 of H10 [SGGG GSGGGGGSGGGG (SEQ ID NO: 27)]. The C-terminal lysine of SEQ ID NO: 15 is optional.

[0258] Biological activity of IL-12 variant / anti-PD1 fusion protein

[0259] In addition to binding to an epitope on PD1, the IL-12 variant / anti-PD1 fusion protein of the present disclosure may also mediate biological activity. That is, the present disclosure includes an isolated IL-12 variant / anti-PD1 fusion protein that specifically binds to PD1 and mediates at least one detectable activity selected from the following:

[0260] (i) specifically binds to human PD1;

[0261] (ii) specifically binds to cynomolgus monkey PD1;

[0262] (iii) Inhibition of tumor growth

[0263] (iv) increase STAT4 phosphorylation;

[0264] (v) increase interferon (IFN)γ expression;

[0265] Without being bound by a particular theory, administering the IL-12 variant / anti-PD1 fusion protein provided herein to a subject can effectively deliver IL-12 to PD1-positive cells [e.g., tumor infiltrating lymphocytes (TIL)] in the tumor microenvironment (TME) with minimal peripheral activity to enhance the anti-tumor activity of TIL and reduce the risk of IL-12 systemic toxicity. In addition, the PD1-positive T cells in the TME are considered to be strong mediators of anti-tumor activity. This may enhance the anti-tumor efficacy of IL-12 in tumors that have not been treated with PD(L)1 (i.e., drug therapy that blocks the interaction between PD1 and PDL1 has not been used before) and PD(L)1-resistant (i.e., drug therapy that blocks the interaction between PD1 and PDL1 has been used before), while reducing systemic immune system activation and the potential toxicity of IL-12. PD1-positive (PD1+) cells in the tumor microenvironment include, for example, CD8-positive (CD8+) T cells, CD4-positive (CD4+) T cells, and regulatory T cells (Treg).

[0266] Similarly, in the non-tumor microenvironment (i.e., peripheral or normal tissue), there are lower abundances of PD1-positive cells, and thus the IL-12 variants / anti-PD1 fusion proteins provided herein result in minimal activity and toxicity due to the attenuation of IL-12 activity in the IL-12 variants and reduced PD1 binding due to the low number of PD1-positive cells.

[0267] In some embodiments, binding of the IL-12 variant / anti-PD1 fusion protein to PD1 promotes inhibition of tumor growth in PD1 / PDL1 therapy-resistant cancer cells [i.e., cancer cells resistant to treatment with one or both of PD1 and PDL1 (collectively referred to as "PD(L)1") inhibitors].

[0268] Binding of IL-12 to IL-12R induces STAT4 phosphorylation (pSTAT4), which in turn upregulates the transcription of type 1 helper T cell ("Th1")-related genes, such as IFNγ, thereby enhancing the functional capacity of T cells. Since phosphorylation of STAT4 is the major activation step of IL-12-induced IL-12 receptor dimerization, pSTAT4 can serve as a receptor-proximal readout of IL-12 activity, while IFNγ can serve as a downstream readout of IL-12 activity.

[0269] Polynucleotides encoding IL-12 variants, anti-PD1 antibodies, fusion proteins and preparation methods

[0270] The present disclosure also provides polynucleotides encoding any IL-12 variant, anti-PD1 antibody or fusion protein provided herein, including portions and modified forms of these molecules. Also included are methods for preparing any IL-12 variant, anti-PD1 antibody and fusion protein provided herein. Polynucleotides can be prepared and proteins expressed by methods known in the art.

[0271] The anti-PD1 antibody of interest (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. Recombinant monoclonal antibodies can be produced in cell culture by cloning antibody genes from B cells in a manner known in the art. See, for example, Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S. Pat. No. 7,314,622.

[0272] In some embodiments, provided herein are polynucleotides comprising sequences encoding one or both of the p35 and p40 subunits of the IL-12 variants provided herein. In some embodiments, provided herein are polynucleotides comprising sequences encoding any one or more polypeptides of the IL-12 variants / anti-PD1 fusion proteins provided herein. In some embodiments, provided herein are polynucleotides comprising sequences encoding one or both of the heavy chain or light chain variable regions of the anti-PD1 antibodies provided herein. The polynucleotides encoding the IL-12 variants, antibodies, or fusion proteins of interest may be maintained in a vector of a host cell, which may then be amplified and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0273] In some embodiments, the disclosure provides a polynucleotide encoding an amino acid sequence of the p35 subunit of an IL-12H10 variant, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 3. In some embodiments, a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 44 encodes the amino acid sequence of SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 44 is shown in Table 7 below.

[0274] In some embodiments, the disclosure provides polynucleotides encoding the amino acid sequence of the p40 subunit of an IL-12H10 variant, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 4. In some embodiments, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 45 encodes the amino acid sequence of SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 45 is shown in Table 7 below.

[0275] In some embodiments, the disclosure provides a polynucleotide encoding the amino acid sequence of VH of anti-PD1 mAb TPP-77658, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 7. In some embodiments, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 46 encodes the amino acid sequence of SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO: 46 is shown in Table 7 below.

[0276] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the VL of the anti-PD1 mAb TPP-77658, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 47 encodes the amino acid sequence of SEQ ID NO: 8. The nucleotide sequence of SEQ ID NO: 47 is shown in Table 7 below.

[0277] In some embodiments, the disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain of the H10658 fusion, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 48 encodes the amino acid sequence of SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 48 is shown in Table 7 below.

[0278] In some embodiments, the disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain-H10 mutant protein p35 fusion of the H10658 fusion, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 49 encodes the amino acid sequence of SEQ ID NO: 25. The nucleotide sequence of SEQ ID NO: 49 is shown in Table 7 below.

[0279] In some embodiments, the disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 light chain of the H10658 fusion, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 50 encodes the amino acid sequence of SEQ ID NO: 6. The nucleotide sequence of SEQ ID NO: 50 is shown in Table 7 below.

[0280] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the H10 mutant protein p40 subunit of the H10658 fusion, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 45 encodes the amino acid sequence of SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 45 is shown in Table 7 below.

[0281] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the p35 subunit of an IL-12H10 variant, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:44.

[0282] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the p40 subunit of an IL-12H10 variant, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:45.

[0283] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of VH of the anti-PD1 mAb TPP-77658, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:46.

[0284] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of VL of the anti-PD1 mAb TPP-77658, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:47.

[0285] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain of the H10658 fusion, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:48.

[0286] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain-H10 mutein p35 fusion of the H10658 fusion, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:49.

[0287] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 light chain of the H10658 fusion, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:50.

[0288] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the H10 mutant protein p40 subunit of the H10658 fusion, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:45.

[0289] Table 7: Nucleotide sequence

[0290]

[0291]

[0292]

[0293] In some embodiments, the present disclosure provides polynucleotides comprising one or more nucleotide sequences encoding any IL-12 variant provided herein. In some embodiments, the present disclosure provides polynucleotides comprising one or more nucleotide sequences encoding IL-12 variant H10, wherein the variant H10 comprises the p35 subunit amino acid sequence of SEQ ID NO: 3 and the p40 subunit amino acid sequence of SEQ ID NO: 4. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding IL-12 variants H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, H25, H30, H31 or H32 as described in Example 1 herein.

[0294] In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding any anti-PD1 antibody provided herein. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 7 and VL comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 17 and VL comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 33 and VL comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO: 6, the heavy chain comprising the amino acid sequence of SEQ ID NO: 5, 51 or 52, wherein the C-terminal lysine of SEQ ID NO: 5, 51 or 52 is optionally present. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO: 16, the heavy chain comprising the amino acid sequence of SEQ ID NO: 15, 53 or 54, wherein the C-terminal lysine of SEQ ID NO: 15, 53 or 54 is optionally present. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 43, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 42, 55 or 56, wherein the C-terminal lysine of SEQ ID NO: 42, 55 or 56 is optionally present.

[0295] In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding any IL-12 variant / anti-PD1 fusion protein provided herein. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an IL-12 variant / anti-PD1 fusion protein, the IL-12 variant / anti-PD1 fusion protein comprising 1) an antibody heavy chain, 2) an antibody heavy chain linked to a p35 subunit of an H10 IL-12 mutant protein; 3) an antibody light chain; and 4) a p40 subunit of an H10 IL-12 mutant protein, wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 5, wherein the antibody heavy chain linked to the p35 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 25, wherein the antibody light chain comprises the amino acid sequence of SEQ ID NO: 6, and wherein the p40 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the present disclosure provides one or more polynucleotides comprising one or more nucleotide sequences encoding an IL-12 variant / anti-PD1 fusion protein, the IL-12 variant / anti-PD1 fusion protein comprising 1) an antibody heavy chain, 2) an antibody heavy chain linked to a p35 subunit of an H10 IL-12 mutant protein; 3) an antibody light chain; and 4) a p40 subunit of an H10 IL-12 mutant protein, wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 15, wherein the antibody heavy chain linked to the p35 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 26, wherein the antibody light chain comprises the amino acid sequence of SEQ ID NO: 16, and wherein the p40 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 4.

[0296] In some embodiments, provided herein are polynucleotides comprising a nucleic acid sequence of an insert fragment of a TPP-77658 heavy chain encoding an H10658 fusion deposited in ATCC with accession number PTA-127517. In some embodiments, provided herein are polynucleotides comprising a nucleic acid sequence of an insert fragment of a TPP-77658 heavy chain-H10 mutant protein p35 fusion polypeptide encoding an H10658 fusion deposited in ATCC with accession number PTA-127518. In some embodiments, provided herein are polynucleotides comprising a nucleic acid sequence of an insert fragment of a TPP-77658 light chain encoding an H10658 fusion deposited in ATCC with accession number PTA-127519. In some embodiments, provided herein are polynucleotides comprising a nucleic acid sequence of an insert fragment of a plasmid deposited in ATCC with accession number PTA-127520 encoding an H10 mutant protein p40 subunit of an H10658 fusion.

[0297] In addition, the present invention also provides a polypeptide comprising an amino acid sequence encoded by a DNA insert fragment of a plasmid deposited in ATCC with accession number PTA-127517 of TPP-77658 heavy chain encoding H10658 fusion. The present invention also provides a polypeptide comprising an amino acid sequence encoded by a DNA insert fragment of a plasmid deposited in ATCC with accession number PTA-127518 of TPP-77658 heavy chain-H10 mutant protein p35 fusion encoding H10658 fusion. The present invention also provides a polypeptide comprising an amino acid sequence encoded by a DNA insert fragment of a plasmid deposited in ATCC with accession number PTA-127519 of TPP-77658 light chain encoding H10658 fusion. The present invention also provides a polypeptide comprising an amino acid sequence encoded by a DNA insert fragment of a plasmid deposited in ATCC with accession number PTA-127520 of H10 mutant protein p40 subunit encoding H10658 fusion.

[0298] In some embodiments, provided herein are anti-PD1 antibodies comprising VH encoded by a portion of a DNA insert of a plasmid deposited with ATCC with accession number PTA-127517 and VL encoded by a portion of a DNA insert of a plasmid deposited with ATCC with accession number PTA-127519. In some embodiments, provided herein are anti-PD1 antibodies comprising a heavy chain encoded by a DNA insert of a plasmid deposited with ATCC with accession number PTA-127517 and a light chain encoded by a DNA insert of a plasmid deposited with ATCC with accession number PTA-127519. In some embodiments, provided herein are anti-PD1 antibodies comprising a heavy chain encoded by a DNA insert of a plasmid deposited with ATCC with accession number PTA-127518 and a light chain encoded by a DNA insert of a plasmid deposited with ATCC with accession number PTA-127519. In some embodiments, provided herein is an IL-12 variant comprising a p35 subunit encoded by a portion of a DNA insert of a plasmid deposited with ATCC with Accession No. PTA-127518 and a p40 subunit encoded by a DNA insert of a plasmid deposited with ATCC with Accession No. PTA-127520.

[0299] It will be appreciated by those of ordinary skill in the art that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides described herein. Some of these polynucleotides have minimal homology with the nucleotide sequences provided herein. Nevertheless, the present invention particularly contemplates polynucleotides that vary due to differences in codon usage. In addition, the alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that change due to one or more mutations such as the deletion, addition or substitution of nucleotides. The resulting mRNA and protein may but may not have a changed structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, or database sequence comparison).

[0300] In one embodiment, the VH and VL domains or full-length HC or LC are encoded by separate polynucleotides. Alternatively, VH and VL, or HC and LC are encoded by a single polynucleotide chain.

[0301] Polynucleotides complementary to any such sequences are also encompassed in the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond one-to-one with DNA molecules, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present in the polynucleotides of the present disclosure, and polynucleotides may, but need not, be connected to other molecules or support materials.

[0302] Polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods or PCR. The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail herein. Those skilled in the art can use the sequence provided herein and commercial DNA synthesizer to produce required DNA sequence.

[0303] In order to prepare polynucleotides using recombinant methods, the polynucleotides comprising the desired sequence can be inserted into a suitable vector, and the vector can then be introduced into a suitable host cell for replication and amplification, as further discussed herein. The polynucleotides can be inserted into a host cell by any means known in the art. Exogenous polynucleotides are introduced to transform cells by direct uptake, endocytosis, transfection, F-conjugation or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as a non-integrated vector (e.g., plasmid) or integrated into the host cell genome.

[0304] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary according to the host cell to be used, useful cloning vectors generally have one or more characteristics, such as i) the ability to self-replicate, ii) a single target for a specific restriction endonuclease, or iii) a marker gene that can be carried to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0305] An expression vector is further provided. An expression vector is typically a reproducible polynucleotide construct containing a polynucleotide according to the present invention. This means that the expression vector must be able to replicate in a host cell as an episome or as a component of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, including viral vectors such as adenovirus, adeno-associated virus, and retrovirus, cosmids, and expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components may typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation start sites, and stop codons, are typically also required.

[0306] The vector containing the polynucleotide of interest can be introduced into the host cell by any of a variety of appropriate methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, when the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0307] The present invention also provides host cells comprising any polynucleotide described herein. Any host cell capable of overexpressing heterologous DNA can be used to separate genes encoding antibodies, polypeptides or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. Also referring to PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., Escherichia coli (E. coli) or Bacillus subtilis (B. subtillis)) and yeast (e.g., Saccharomyces cerevisiae (S. cerevisae), Schizosaccharomyces pombe (S. pombe); or Kluyveromyces lactis (K. lactis)).

[0308] In addition, any number of commercial and non-commercially available cell lines expressing polypeptides or proteins can be used according to the present invention. Those skilled in the art will appreciate that different cell lines may have different nutritional requirements or may require different culture conditions to achieve optimal growth and polypeptide or protein expression, and will be able to modify the conditions as needed.

[0309] Pharmaceutical composition

[0310] In another embodiment, the present invention includes pharmaceutical compositions.

[0311] A "pharmaceutical composition" refers to a mixture of an IL-12 variant, an anti-PD1 antibody or a fusion protein of the present invention and one or more excipients.

[0312] The pharmaceutical compositions of the present invention can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, and lyophilized powders. The form depends on the intended mode of administration and therapeutic application.

[0313] Other excipients and modes of administration known in the pharmaceutical field may also be used. The pharmaceutical compositions of the present invention may be prepared by any well-known pharmaceutical techniques, such as effective formulation and administration procedures. The above considerations regarding effective formulation and administration procedures are well known in the art and described in standard textbooks. The formulation of drugs is described, for example, in the following documents: Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0314] Acceptable excipients are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0315] Treatment, diagnosis and other methods

[0316] The IL-12 variants, anti-PD1 antibodies and IL-12 variant / anti-PD1 fusion proteins of the present invention can be used in a variety of applications, including but not limited to as medicaments, in therapeutic treatment methods and in diagnostic methods.

[0317] In some embodiments, the IL-12 variants and IL-12 variant / anti-PD1 fusion proteins provided herein can be used to treat any condition in which a subject would benefit from increased IL-12 activity. The IL-12 variants and IL-12 variant / anti-PD1 fusion proteins provided herein are particularly useful in situations in which it is desired to provide IL-12 activity to a subject in a strictly controlled or limited manner.

[0318] In one aspect, the present invention provides a method for treating cancer. In some embodiments, the method for treating cancer in a subject comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising any of the IL-12 variants, antibodies, or fusion proteins described herein. In some embodiments, a method for treating cancer in a subject is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising an IL-12 variant, antibody, or fusion protein provided herein.

[0319] In some embodiments, provided herein is a method of inhibiting the growth of PD1 / PD-L1 therapy-resistant cancer cells in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising an IL-12 variant or IL-12 variant / anti-PD1 fusion protein provided herein.

[0320] In some embodiments, provided herein is a method for promoting CD8-positive (+) T cell infiltration in a subject's tumor microenvironment, comprising administering to a subject in need thereof an effective amount of a composition comprising an IL-12 variant or IL-12 variant / anti-PD1 fusion protein provided herein.

[0321] In some embodiments, provided herein is a method for promoting STAT4 phosphorylation in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising an IL-12 variant or an IL-12 variant / anti-PD1 fusion protein provided herein.

[0322] In some embodiments, provided herein is a method for promoting interferon gamma (IFNg) production in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising an IL-12 variant or IL-12 variant / anti-PD1 fusion protein provided herein.

[0323] On the other hand, the present invention also provides the IL-12 variants, IL-12 variants / anti-PD1 fusion proteins or related pharmaceutical compositions described herein for use in the method for treating cancer. The present invention also provides the use of the IL-12 variants or IL-12 variants / anti-PD1 fusion proteins described herein in the production of drugs for treating cancer.

[0324] In some embodiments, cancers that can be treated with IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins provided herein include, for example, solid tumors or liquid tumors. In some embodiments, solid tumors treated with IL-12 variants provided herein include, for example, non-small cell lung cancer (NSCLC), ovarian cancer, renal cell carcinoma (RCC), colorectal cancer (CRC), and hepatocellular carcinoma (HCC). In some embodiments, treatable cancers include one or more of the following: bladder cancer, breast cancer, clear cell renal carcinoma, head / neck squamous cell carcinoma (HNSCC) [head and neck squamous cell carcinoma (SCCHN)], lung squamous cell carcinoma, lung adenocarcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma (RCC), small cell lung cancer (SCLC), triple-negative breast cancer, urothelial carcinoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic Myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL), endometrial cancer, B-cell acute lymphoblastic leukemia, colorectal cancer (CRC), glioblastoma, uterine cancer, cervical cancer, penile cancer, gastric cancer (GC) and non-melanoma skin cancer. In some embodiments, cancers that can be treated with the IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins provided herein include, for example, NSCLC previously treated with platinum therapy and / or a checkpoint inhibitor (e.g., a PD(L)1 inhibitor), RCC previously treated with a tyrosine kinase inhibitor and / or a checkpoint inhibitor (e.g., a PD(L)1 inhibitor), ovarian cancer, microsatellite stable (MSS) CRC, hepatocellular carcinoma (HCC), or bladder cancer.

[0325] Administration and Dosage

[0326] Typically, the IL-12 variants, anti-PD1 antibodies or IL-12 variant / anti-PD1 fusion proteins of the invention are administered in an amount effective to treat the conditions described herein. The molecules of the invention can be administered as the molecule itself, or as a pharmaceutical composition containing the molecule.

[0327] The molecules of the invention are administered by any appropriate route in the form of a pharmaceutical composition suitable for that route and in a dose effective for the intended treatment.

[0328] In some embodiments, the antibody can be administered parenterally, for example, directly into the bloodstream, into a muscle, or into an internal organ. Suitable modes for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous administration. Suitable devices for parenteral administration include needle (including microneedle) syringes, needle-free syringes, and infusion techniques. In some embodiments, IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins provided herein are administered subcutaneously (SC). In some embodiments, IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins provided herein are administered intravenously (IV).

[0329] The dosage regimen of the antibody of the present invention or the composition containing the antibody is based on a variety of factors, including the type, age, weight, sex and medical condition of the subject; the severity of the condition; the route of administration; and the activity of the specific antibody used. Therefore, the dosage regimen may vary greatly. In one embodiment, the total daily dose of the antibody of the present invention is generally about 0.01 to about 100 mg / kg (i.e., mg of the antibody of the present invention per kg of body weight) for the treatment of the specified conditions discussed herein. In another embodiment, the total daily dose of the antibody of the present invention is about 0.1 to about 20 mg / kg, and in another embodiment, about 0.5 to about 10 mg / kg.

[0330] In some embodiments, the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein provided herein is administered once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once a month (Q1M), once every two months (Q2M), or once every three months (Q3M).

[0331] In some embodiments, the IL-12 variant / anti-PD1 fusion protein is provided at a dose of about 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg.

[0332] In some embodiments, the IL-12 variant / anti-PD1 fusion protein is provided IV Q2W at a dose of 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg or 15 mg / kg. In some embodiments, the IL-12 variant / anti-PD1 fusion protein is provided IV Q3W at a dose of 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg or 15 mg / kg. In some embodiments, the IL-12 variant / anti-PD1 fusion protein is provided SC Q2W at a dose of 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg or 15 mg / kg. In some embodiments, the IL-12 variant / anti-PD1 fusion protein is provided SC Q3W at a dose of 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg or 15 mg / kg.In some embodiments, the IL-12 variant / anti-PD1 fusion protein provided at the above doses is the H10868 fusion or the H10658 fusion described in Example 3 herein.

[0333] Toxicity and efficacy of the prophylactic and / or therapeutic regimens of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD 50 / ED 50 Preferred are prophylactic and / or therapeutic agents that exhibit large therapeutic indices.

[0334] Co-administration

[0335] The IL-12 variants, anti-PD1 antibodies and IL-12 variant / anti-PD1 fusion proteins provided herein can be used alone or in combination with one or more other therapeutic agents. Provided herein are any uses, methods or compositions as defined herein, wherein the IL-12 variants, anti-PD1 antibodies or IL-12 variant / anti-PD1 fusion proteins of the present invention are used in combination with one or more other therapeutic agents discussed herein.

[0336] Administration of two or more agents "in combination" means that all agents are administered in sufficient time to affect the treatment of the subject. The two or more agents may be administered simultaneously or sequentially. Additionally, simultaneous administration may be performed by mixing the agents prior to administration or by administering the agents in separate dosage forms at the same time point but at the same or different administration sites.

[0337] In some embodiments, the IL-12 variants, anti-PD1 antibodies or IL-12 variants / anti-PD1 fusion proteins provided herein can be administered in combination with the administration of one or more additional therapeutic agents. Optionally, the additional therapeutic agent may include an additional anticancer agent. These include, but are not limited to, the administration of biological therapeutics and / or chemotherapeutics, such as, but not limited to, vaccines, CAR-T cell-based therapies, radiotherapy, cytokine therapy, CD3 bispecific antibodies, other immunosuppressive pathway inhibitors, angiogenesis inhibitors, T cell activators, metabolic pathway inhibitors, mTOR inhibitors, adenosine pathway inhibitors, tyrosine kinase inhibitors including, but not limited to, Inlyta, ALK inhibitors and sunitinib, BRAF inhibitors, epigenetic modifiers, IDO1 inhibitors, JAK inhibitors, STAT inhibitors, cyclin-dependent stimulants. Enzyme inhibitors, biotherapeutics (including but not limited to antibodies to VEGF, VEGFR, EGFR, Her2 / neu, other growth factor receptors, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, TIGIT and ICOS), immunogenic agents (e.g., attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF, and cells transfected with genes encoding immunostimulatory cytokines such as but not limited to GM-CSF).

[0338] Examples of biotherapeutics include therapeutic antibodies, immunomodulators, and therapeutic immune cells.

[0339] Therapeutic antibodies can be specific for a variety of different antigens. For example, therapeutic antibodies can be directed to tumor-associated antigens so that the combination of antibodies and antigens promotes the death of cells expressing the antigen. In other examples, therapeutic antibodies can be directed to antigens on immune cells (such as PD1) so that the combination of antibodies prevents the activity of cells expressing antigens from being downregulated (and thereby promotes the activity of cells expressing antigens). In another example, therapeutic activity can be directed to antigens so that the combination of antibodies and antigens stimulates target molecules containing the antigen (i.e., the antibody is an agonist antibody) to promote the desired activity. In some cases, therapeutic antibodies can work through a variety of different mechanisms (for example, they can i) promote the death of cells expressing antigens, and ii) prevent antigens from causing immune cell activity to be downregulated in contact with cells expressing antigens).

[0340] Therapeutic antibodies can be directed against, for example, the antigens listed below. For some antigens, exemplary antibodies against the antigen are also included below (in brackets / parentheses after the antigen). The following antigens may also be referred to as "target antigens" and the like in this article. Target antigens for therapeutic antibodies herein include, for example: 4-1BB (e.g., utomilumab); 5T4; A33; α-folate receptor 1 (e.g., mirvetuximab soravtansine); Alk-1; BCMA [e.g., PF-06863135 (see US9969809)]; BTN1A1 (e.g., see WO2018222689); CA-125 (e.g., abagovomab); Carboanhydrase IX; CCR2; CCR4 (e.g., mogamulizumab); CCR5 (e.g., leronlimab); CCR8; CD3 [e.g., blinatumomab (CD3 / CD19 bispecific), PF-06671008 (CD3 / P-cadherin bispecific), PF-06863135 (CD3 / BCMA bispecific), CD19 (e.g., blinatumomab, MOR208); CD20 (e.g., ibritumomab tiuxetan tiuxetan, obinutuzumab, ofatumumab, rituximab, ublituximab); CD22 (inotuzumab bozogamicin, moxetumomab pasudotox); CD25; CD28; CD30 (e.g., brentuximab vedotin); CD33 (e.g., gemtuzumab tuzumab); ozogamicin); CD38 (e.g., daratumumab, isatuximab), CD40; CD-40L; CD44v6; CD47; CD52 (e.g., alemtuzumab); CD63; CD79 (e.g., polatuzumab vedotin); CD80; CD123; CD276 / B7-H3 (e.g., omburtamab); CDH17; CEA; ClhCG; CTLA-4 (e.g., ipilimumab, tremelimumab), CXCR4; desmoglein 4;DLL3 (e.g., rovalpituzumab tesirine); DLL4; E-cadherin; EDA; EDB; EFNA4; EGFR (e.g., cetuximab, depatuxizumab mafodotin, necitumumab, panitumumab); EGFRvIII; Endosialin; EpCAM (e.g., oportuzumab monatox); FAP; fetal acetylcholine receptor; FLT3 (e.g., see WO2018 / 220584); GD2 (e.g., dinutuximab, 3F8); GD3; GITR; GloboH; GM1; GM2; GUCY2C (e.g., PF-07062119); HER2 / neu [e.g., margetuximab, pertuzumab, trastuzumab; ado-trastuzumab emtansine, trastuzumab duocarmazine, PF-06804103 (see US8828401)]; HER3; HER4; ICOS; IL-10; ITG-AvB6; LAG-3 (e.g., relatlimab); Lewis-Y; LG; Ly-6; M-CSF [e.g., PD-0360324 (see US7326414)]; MCSP; mesothelin; MUC1; MUC2; MUC3; MUC4; MUC5AC; MUC5B; MUC7; MUC16; Notch1; Notch3; Nectin-4 (e.g., enfortumab vedotin)); OX40 [e.g. PF-04518600 (see US7960515)]; P-cadherin [e.g. PF-06671008 (see WO2016 / 001810)]; PCDHB2;PD1 [e.g., BCD-100, camrelizumab, cemiplimab, genolimzumab (CBT-501), MEDI0680, nivolumab, pembrolizumab, RN888 (see WO2016 / 092419), sintilimab, spartalizumab, STI-A1110, tislelizumab, TSR-042]; PD-L1 (e.g., atezolizumab, durvalumab durvalumab), BMS-936559 (MDX-1105), or LY3300054); PDGFRA (e.g., olaratumab); plasma cell antigen; PolySA; PSCA; PSMA; PTK7 [e.g., PF-06647020 (see US9409995)]; Ror1; SAS; SCRx6; SLAMF7 (e.g., elotuzumab); SHH; SIRPa (e.g., ED9, Effi-DEM); STEAP; TGF-β; TIGIT; TIM-3; TMPRSS3; TNF-α precursor; TROP-2 (e.g., sacituzumab govitecan); TSPAN8; VEGF (e.g., bevacizumab, brolucizumab); VEGFR1 (e.g., ranibizumab); VEGFR2 (e.g., ramucirumab, ranibizumab); Wue-1. ;

[0341] The therapeutic antibodies administered in combination with the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein may have any suitable form. For example, the therapeutic antibodies may have any form described elsewhere herein. In some embodiments, the therapeutic antibodies may be naked antibodies. In some embodiments, the therapeutic antibodies may be linked to drugs or other agents (also referred to as "antibody-drug conjugates" (ADC)). In some embodiments, therapeutic antibodies directed against specific antigens may be incorporated into multispecific antibodies (e.g., bispecific antibodies).

[0342] In some embodiments, the IL-12 variants, anti-PD1 antibodies or IL-12 variants / anti-PD1 fusion proteins provided herein can be administered in combination with pattern recognition receptor (PRR) agonists, immunostimulatory cytokines and cancer vaccines. There are many categories of PRR molecules, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs) and interferon gene stimulators (STING) proteins. Other PRRs include, for example, DNA-dependent IFN regulatory factor activators (DAI) and melanoma deficiency factor 2 (AIM2). In some embodiments, the IL-12 variants, anti-PD1 antibodies or IL-12 variants / anti-PD1 fusion proteins provided herein can be administered in combination with TLR agonists (e.g., TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9 agonists).

[0343] Examples of immunostimulatory cytokines that can be used in the methods of treatment, medicaments and uses of the invention include GM-CSF, G-CSF, IFN-α, IFN-γ; IL-2 (e.g., denileukin difitox), IL-6, IL-7, IL-11, IL-15, IL-18, IL-21 and TNF-α.

[0344] Examples of cancer vaccines that can be used in the treatment methods, medicaments and uses of the present invention include, for example, sipuleucel-T and talimogene laherparepvec (T-VEC).

[0345] Examples of immune cell therapies that can be used in the treatment methods, medicaments, and uses of the present invention include, for example, tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor T cells (CAR-T cells).

[0346] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylethyleneimines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin (synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma and calicheamicin phi, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and the neocarcinogen chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycins, authramycins, azaserines, bleomycins, cactinomycin, karubicin carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolino doxorubicin and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folic acid; acetylacetone; aldophosphamide glycosides; aminolevulinic acid acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazine; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine;trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; Mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are anti-hormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, megestrolacetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; KRAS inhibitors; MCT4 inhibitors; MAT2a inhibitors; tyrosine kinase inhibitors, For example, sunitinib, axitinib; alk / c-Met / ROS inhibitors, such as crizotinib, lorlatinib; mTOR inhibitors, such as temsirolimus, gedatolisib; src / abl inhibitors, such as bosutinib; cyclin-dependent kinase (CDK) inhibitors, such as palbociclib, PF-06873600; erb inhibitors, such as dacomitinib; PARP inhibitors, such as talazoparib; SMO inhibitors, such as glasdegib, PF-5274857; EGFR T790M inhibitors, such as PF-06747775; EZH2 inhibitors, such as PF-06821497; PRMT5 inhibitors, such as PF-06939999; TGFRβr1 inhibitors, such as PF-06952229;and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. In specific embodiments, such additional therapeutic agents are bevacizumab, cetuximab, sirolimus, panitumumab, 5-fluorouracil (5-FU), capecitabine, tivozanib, irinotecan, oxaliplatin, cisplatin, trifluridine, tipiracil, leucovorin, gemcitabine, regorafinib or erlotinib hydrochloride. ;

[0347] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins provided herein are administered in combination with PD1 or PDL1 inhibitors. PD1 and PDL1 inhibitors are collectively referred to herein as "PD(L)1" inhibitors. In some embodiments, the PD(L)1 inhibitor is sasanlimab.

[0348] In some embodiments, the PD(L)1 inhibitor is an anti-PD1 or anti-PD-L1 antibody. These antibodies bind to PD1 or PDL1 and block the interaction between PD1 and PDL1. Examples of PD(L)1 inhibitors that can be used in the methods, drugs, and uses of the present invention include, for example, saxanthlimab (also known as RN888, an anti-PD1 IgG4 monoclonal antibody), pembrolizumab (also known as MK-3475, an anti-PD1 IgG4 monoclonal antibody), nivolumab (also known as BMS-936558 or MDX1106, an anti-PD1 IgG4 monoclonal antibody), cemiplimab (also known as REGN-2810, an anti-PD1 antibody), atezolizumab (also known as MPDL3280A, an IgG1 engineered anti-PDL1 antibody), BMS-936559 (a fully human anti-PDL1 IgG4 monoclonal antibody), MEDI4736 (also known as durvalumab, an engineered IgG1κ anti-PDL1 monoclonal antibody with triple mutations in the Fc domain to eliminate antibody-dependent, cell-mediated cytotoxic activity). Additional exemplary PD(L)1 inhibitors that can be used in the methods, drugs, and uses of the present invention include SHR1210 (anti-PD1 antibody), KN035 (anti-PD1 antibody), IBI308 (anti-PD1 antibody), PDR001 (anti-PD1 antibody), BGB-A317 (anti-PD1 antibody), BCD-100 (anti-PD1 antibody), JS001 (anti-PD1 antibody). In some embodiments, the PD(L)1 inhibitor is a small molecule PD1 or PDL1 antagonist (e.g., CA-170), as described in Yang et al Med. Res. Rev. (2019), 39, pp265-301.

[0349] In some cases, it is advantageous to combine the IL-12 variants / anti-PD1 fusion proteins provided herein with anti-PD1 antibodies that bind to different epitopes on PD1 rather than with anti-PD1 antibodies of IL-12 variants / anti-PD1 fusion proteins. For example, some anti-PD1 antibodies provided herein bind to epitopes on PD1 such that the antibodies do not block the interaction between PD1 and PDL1. In contrast, most or all of the anti-PD1 antibodies previously approved for therapeutic use bind to epitopes on PD1 such that the antibodies inhibit the interaction between PD1 and PDL1.

[0350] Anti-PD1 antibodies that do not block the interaction between PD1 and PDL1 can be used to target IL-12 variant / anti-PD1 fusion proteins to PD1-expressing cells, such as T cells in the tumor microenvironment.

[0351] Therefore, in some embodiments, provided herein is a combination therapy comprising 1) an IL-12 variant / anti-PD1 fusion protein provided herein, wherein the anti-PD1 antibody of the fusion protein does not block the interaction between PD1 and PDL1, and 2) a PD(L)1 inhibitor, wherein the PD(L)1 inhibitor blocks the interaction between PD1 and PDL1. In some embodiments, the PD(L)1 inhibitor is an anti-PD1 antibody that inhibits the interaction between PD1 and PDL1. In some embodiments, the PD(L)1 inhibitor is an anti-PDL1 antibody that inhibits the interaction between PD1 and PDL1.

[0352] In some embodiments, the IL-12 variants, anti-PD1 antibodies or IL-12 variants / anti-PD1 fusion proteins provided herein are administered in combination with VEGF or VEGF receptor (VEGFR) inhibitors. VEGF and VEGFR inhibitors are collectively referred to herein as "VEGF (R)" inhibitors. VEGF (R) inhibitors include agents that bind any VEGF subtype (e.g., VEGF-A, VEGF-C, and VEGF-D) and VEGFR subtypes (e.g., VEGFR1, VEGFR2, and VEGFR3). In some embodiments, the VEGF (R) inhibitor is axitinib or bevacizumab.

[0353] In some embodiments, VEGF (R) inhibitors are anti-VEGF or anti-VEGFR antibodies. It binds to VEGF or VEGFR and blocks the interaction between VEGF and VEGFR and / or inhibits the activity of VEGFR. Anti-VEGF (R) antibodies include, for example, bevacizumab, ramucirumab, and ranibizumab. In some embodiments, VEGF (R) inhibitors are small molecule agents that bind to VEGF or VEGFR and inhibit VEGFR activity. Small molecule VEGF(R) inhibitors include, for example, apatinib, axitinib, cabozantinib, lapatinib, lenvatinib, nintedanib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib and vandetanib.

[0354] In some embodiments, the IL-12 variants, anti-PD1 antibodies or IL-12 variants / anti-PD1 fusion proteins provided herein can be co-administered with other pharmaceutical treatments, or can be sequentially administered with a time interval ranging from a few minutes to a few weeks before or after the other pharmaceutical treatments. In embodiments where other agents and / or proteins or polynucleotides are administered separately, it is generally ensured that a considerable period of time does not pass between each delivery, so that the agents and compositions of the present invention can still exert a favorable combined effect on the subject. In this case, it is expected that the two methods can be administered within about 12 to 24 hours of each other, and more preferably within about 6 to 12 hours of each other. However, in some cases, it may be necessary to significantly extend the administration time, wherein a few days (2, 3, 4, 5, 6 or 7 days) to a few weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) are spaced between each administration.

[0355] In some embodiments, the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein is combined with a treatment regimen further comprising a conventional therapy selected from the group consisting of surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative care.

[0356] Reagent test kit

[0357] Another aspect of the present invention provides a kit comprising an IL-12 variant, an anti-PD1 antibody or an IL-12 variant / anti-PD1 fusion protein provided herein. In addition to the IL-12 variant, the anti-PD1 antibody or the IL-12 variant / anti-PD1 fusion protein, the kit may also include one or more diagnostic agents or therapeutic agents. The kit may also include instructions for use in diagnostic or therapeutic methods. In some embodiments, the kit includes an antibody or a pharmaceutical composition thereof and a diagnostic agent. In other embodiments, the kit includes an antibody or a pharmaceutical composition thereof and one or more therapeutic agents, such as a PD(L)1 inhibitor (e.g., a blocking anti-PD1 antibody).

[0358] In yet another embodiment, the present invention includes a kit suitable for implementing the methods of treatment described herein. In one embodiment, the kit contains a first dosage form comprising one or more of the IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins of the present invention in an amount sufficient to perform the method of the present invention. In another embodiment, the kit comprises one or more of the IL-12 variants, anti-PD1 antibodies, or IL-12 variants / anti-PD1 fusion proteins of the present invention in an amount sufficient to perform the method of the present invention, and at least a first container for the first dose and a second container for the second dose.

[0359] Biological Deposit

[0360] Representative materials of the present invention were deposited on February 7, 2023 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA. The vector "HC_H10658" with ATCC accession number PTA-127517 contains a DNA insert of the TPP-77658 heavy chain encoding the H10658 fusion. The vector "HCp35_H10658" with ATCC accession number PTA-127518 contains a DNA insert of the TPP-77658 heavy chain-H10 mutant protein p35 fusion encoding the H10658 fusion. The vector "LC_H10658" with ATCC accession number PTA-127519 contains a DNA insert of the TPP-77658 light chain encoding the H10658 fusion. The vector "p40_H10658" with ATCC accession number PTA-127520 contains a DNA insert encoding the H10 mutant protein p40 subunit of the H10658 fusion. These are summarized in Table 8 below.

[0361] Table 8: ATCC deposit information

[0362]

[0363] The deposit is made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (Budapest Treaty) and its regulations. This ensures that the deposit remains a viable culture for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer and ATCC that ensures that the culture progeny of the deposit will be permanently and unrestrictedly available to the public upon the issuance of the relevant U.S. patent or the opening of any U.S. or foreign patent application to the public (whichever comes first), and that the progeny can be obtained by the Commissioner of the United States Patent and Trademark under 35 U.S.C. Section 122 and persons determined by the Commissioner under his rules (including 37 C.FR Section 1.14, with particular reference to 886 OG 638).

[0364] The assignee of this application agrees that if a culture of the deposited material dies or is lost or destroyed when grown under appropriate conditions; such material will be promptly replaced with other identical material upon notification. The availability of the deposited material shall not be construed as a license to practice the invention in violation of the rights granted by any government under its patent laws.

[0365] The contents of U.S. Provisional Patent Application No. 63 / 353,241, filed on June 17, 2022, and U.S. Provisional Patent Application No. 63 / 496,545, filed on April 17, 2023, are incorporated herein by reference for all purposes.

[0366] Sequence Overview

[0367] The sequences provided in this application are summarized in Table 9 below.

[0368] Table 9

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377] The foregoing description and the following examples describe in detail certain specific embodiments of the present disclosure and describe the best mode contemplated by the inventors. However, it should be understood that no matter how detailed the foregoing is presented in text, the present disclosure can be practiced in many ways, and the present disclosure should be interpreted according to the appended claims and any equivalents thereof.

[0378] Although the disclosed teachings have been described with reference to various applications, methods, kits and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein and the disclosures claimed below. The following examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the present teachings have been described according to these exemplary embodiments, it will be readily appreciated by those skilled in the art that multiple changes and modifications to these exemplary embodiments are possible without excessive experimentation. All of these changes and modifications are within the scope of the current teachings. Example

[0379] In order to better understand the present invention, the following examples are given. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0380] Example 1: IL-12 variants

[0381] The goal of this experiment was to generate human IL-12 variants (also referred to as IL-12 muteins or "mutants") that have attenuated IL-12 activity relative to wild-type human IL-12.

[0382] A group of IL-12 variants were designed, wherein mutations were engineered into one or both of the p35 and p40 subunits of human IL-12. IL-12 variants are described in Table 10. In Table 10, mutation positions are numbered based on the amino acid sequences of mature human p35 and p40 proteins (SEQ ID NO: 1 and SEQ ID NO: 2, respectively). As shown in Table 10, the mutations are located at one or more of the F39, I52 and Y167 positions of the p35 subunit, and one or more of the D93 and K85 positions of the p40 subunit. These amino acid positions are expected to be located at the interface between IL12 and the IL12 receptor.

[0383] Table 10

[0384]

[0385]

[0386] Fusion proteins were prepared in which the different IL-12 mutant proteins shown in Table 10 were linked to the platform anti-human PD1 antibody. The activities of the different IL-12 mutant proteins / anti-PD1 fusion proteins were then evaluated.

[0387] First, the activity of IL-12 mutein / anti-PD1 fusion protein was evaluated using an IL-12 receptor positive (IL12R+), human PD1 negative pSTAT4 reporter cell line. The half maximal effective concentration (EC 50 ) (evaluating STAT4 phosphorylation) are shown in the "EC50 hIL12R+ cells" column of Table 10. The EC 50 Supplied in micrograms per milliliter (μg / ml). Low EC 50 Values ​​indicate higher EC 50 Values ​​have higher activity.

[0388] Next, the activity of the IL-12 mutein / anti-PD1 fusion protein was assessed using an IL-12 receptor positive (IL12R+), human PD1 positive pSTAT4 reporter cell line. The half maximal effective concentration (EC50 hIL12R+PD1+ cells) of the fusion protein using this cell line is shown in the "EC50 hIL12R+PD1+ cells" column of Table 10. 50) (assessing STAT4 phosphorylation). This data provides information about the PD1-induced rescue of IL-12 mutein activity of each fusion protein (eg, by comparing the activity of each fusion protein between PD1-negative and PD1-positive cell lines).

[0389] As shown in Table 10, various IL-12 mutant proteins / anti-PD1 fusion proteins have low activity in hIL12R+, PD1-negative cells, but have higher activity in hIL12R+, PD1-positive cells (e.g., mutant protein H10). Therefore, these fusion proteins have targeted IL-12 activity on PD1-positive cells.

[0390] As further shown in Table 10, the mutant protein "H10" has an undetermined but lower activity against PD1-negative cells (EC 50 greater than 3000 μg / ml; the highest concentration tested), but had higher activity against PD1-positive cells (EC 50 is 686μg / ml).

[0391] Based on multiple favorable features identified in these assays, the H10 mutant protein was selected for further development. First, as described above, the H10 mutant protein linked to the anti-PD1 antibody has low activity against PD1-negative cells. The lack of activity against PD1-negative cells may limit the number of cells that can be effectively stimulated by the H10 IL-12 mutant protein, and therefore may reduce toxicity associated with IL-12 activity (e.g., overstimulation of immune responses mediated by IL-12). Second, although the H10 mutant protein linked to the anti-PD1 antibody has low activity against PD1-negative cells, the H10 IL-12 mutant protein / anti-PD1 fusion protein still has detectable activity against PD1-positive cells. Therefore, it is assumed that the H10 IL-12 mutant protein / anti-PD1 fusion protein is still active in an environment rich in PD1-positive cells, such as the tumor microenvironment (TME). Third, evaluation of the physiological properties of the H10 mutant protein, such as stability and predicted immunogenicity, indicated that the H10 mutant protein possessed more desirable molecular properties than other IL12 mutant proteins and also had biased activity against PD1-positive cells compared with PD1-negative cells when linked to anti-PD1 antibodies.

[0392] The H10 IL12 mutant protein contains the mature p35 amino acid sequence SEQ ID NO: 3 and the mature p40 amino acid sequence SEQ ID NO: 4. As shown in SEQ ID NO: 3 below, the H10 p35 subunit contains the mutation Y167A (underlined), and as shown in SEQ ID NO: 4 below, the H10 p40 subunit contains the mutation D93L (underlined) and the heparin binding site mutation GGG (underlined). For the heparin binding site mutation, the amino acid sequence KSKREKK (SEQ ID NO: 30) in the wild-type IL-12 p40 sequence (amino acids 258-264 in SEQ ID ID: 4) is mutated and truncated to the sequence "GGG". It is known that wild-type IL-12 binds to heparin and heparan sulfate (Hasan M, et al. J Immunol. 1999 Jan 15; 162 (2): 1064-70); mutation of the sequence KSKREKK (SEQ ID NO: 30) in wild-type IL-12p40 to GGG reduces the affinity of IL-12p40 for heparin and heparan sulfate. It is hypothesized that binding of IL-12 to heparin may enhance IL-12 function (e.g., by retaining IL-12 near the site of secretion to maintain high local cytokine concentrations). In addition, the heparin binding site in IL-12 is protease sensitive; therefore, removal of this site reduces the protease sensitivity of the H10 mutant protein.

[0393] H10 mutant protein p35:

[0394] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDF A KTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:3)

[0395] H10 mutant protein p40:

[0396] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWST LILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQG GGG DRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:4)

[0397] Example 2: Non-blocking anti-PD1 antibodies

[0398] A multi-step approach was used to generate and select novel non-blocking anti-human PD1 (hPD1) antibodies. The anti-hPD1 antibodies generated included clones GBT-PD1-0009, GBT-PD1-0013, and GBT-PD1-0017.

[0399] Non-blocking anti-hPD1 antibodies are antibodies that do not bind to the same epitope on hPD1 that is bound by the hPD1 ligand human PDL1 (hPDL1). Non-blocking anti-hPD1 antibodies do not prevent the binding of hPDL1 to hPD1. In addition, non-blocking antibodies may not prevent the binding of many currently available therapeutic antagonist anti-hPD1 antibodies (e.g., pembrolizumab, cemiplimab, nivolumab, etc.) to PD1, which interfere with the binding of hPDL1 to hPD1.

[0400] To assess whether various antibodies can bind to hPD1 simultaneously, sandwich assays were performed in which 2 different antibodies were incubated simultaneously with hPD1. In addition, in some assays, antibodies were incubated with fusion molecules containing hPDL1 covalently linked to the Fc domain of the antibody (hPDL1-Fc) to assess whether the respective antibodies can bind to hPD1 simultaneously with hPDL1.

[0401] Table 11 shows the results of the sandwich assay. In Table 11, if the paired antibodies listed in the corresponding columns and rows can bind to hPD1 simultaneously (i.e., there is no competition between the antibodies for binding to hPD1), "Y" is listed in the corresponding position in the table, or if there is competition between the antibodies for binding to hPD1, "N" is listed. For example, as shown in Table 11, when nivolumab, pembrolizumab, or hPDL1 is present in each of these positions in the table (indicated by "N"), the binding of cimiprilimumab to hPD1 is interfered. In contrast, in the presence of GBT-PD1-0009, GBT-PD1-0013, or GBT-PD1-0017, the binding of cimiprilimumab to hPD1 is not interfered.

[0402] Table 11

[0403]

[0404] For each of GBT-PD1-0009, GBT-PD1-0013, or GBT-PD1-0017, the binding of the corresponding antibody to PD1 was not interfered with by hPDL1, cemiprizumab, nivolumab, or pembrolizumab, indicating that GBT-PD1-0009, GBT-PD1-0013, and GBT-PD1-0017 do not bind to PD1 at the position bound by hPDL1, or do not bind to the epitope bound by cemiprizumab, nivolumab, or pembrolizumab.

[0405] To verify the non-blocking nature of GBT-PD1-0013, the X-ray crystal structure of GBT-PD1-0013 in complex with the human PD1 extracellular domain (ECD) was elucidated. The crystal structure shows that GBT-PD1-0013 binds to the human PD1 ECD in a 1:1 stoichiometry at a site different from the natural ligand PD-L1, as determined by alignment of related complex structures.

[0406] The simultaneous binding of a blocking anti-human PD1 antibody ["PD1(B)"] and a non-blocking anti-PD1 antibody GBT-PD1-0013 was evaluated by flow cytometry-based readout using the BW5147.3 (T lymphocyte) cell line overexpressing human PD1. To enable flow cytometry-based readout, PD1(B) and GBT-PD1-0013 were conjugated to fluorescent dyes (AlexaFluor488 and AlexaFluor647, respectively). A fixed number of cells were incubated with: i) a single antibody to evaluate maximal individual binding, or ii) a 1:1 mixture of antibodies to evaluate simultaneous binding of the antibodies. An isotype control antibody was used as a negative control for binding to PD1. The results are shown in Figure 1A and 1BIn these figures, GBT-PD1-0013 is referred to as “PD1(NB)” and the blocking anti-PD1 antibody is referred to as “PD1(B)”. Figure 1A and Figure 1B It was shown that the binding of GBT-PD1-0013 to cells expressing PD1 was only minimally affected by the presence of PD1 (B), and vice versa. Figure 1A It was shown that the binding of GBT-PD1-0013 to cells expressing PD1 was only minimally affected by the presence of PD1(B) (i.e., when GBT-PD1-0013 and PD1(B) were incubated with cells expressing PD1, the percentage of cells expressing PD1 bound by GBT-PD1-0013 was only slightly decreased compared to when GBT-PD1-0013 alone was incubated with cells expressing PD1). Similarly, Figure 1B It was shown that the presence of GBT-PD1-0013 had only a minimal effect on the binding of PD1 (B) to cells expressing PD1.

[0407] Clone GBT-PD1-0013 was modified to generate a series of related non-blocking anti-hPD1 antibodies with different affinities for hPD1 and also with reduced predicted immunogenicity (based on a lower number of predicted T cell epitopes) compared to the parent GBT-PD1-0013 antibody. Table 12 provides the binding characteristics of the different clones to hPD1.

[0408] Table 12

[0409]

[0410] Example 3: IL-12 mutant protein-non-blocking anti-PD1 antibody fusion protein

[0411] This example describes the preparation and properties of fusion proteins combining IL-12H10 muteins (Example 1) with various non-blocking anti-PD1 antibodies (Example 2).

[0412] A variety of non-blocking anti-PD1 antibodies as described in Example 2 were covalently linked to the IL-12 mutant protein H10 via a serine-glycine linker. Specifically, the p35 subunit of the IL-12H10 mutant protein was linked to the C-terminus of one heavy chain of the antibody via a linker having the amino acid sequence SGGGGSGGGGGSGGGG (SEQ ID NO: 27). (The p40 subunit of the IL-12H10 mutant protein binds to the antibody through its interaction with the p35 subunit). A general schematic diagram of the fusion protein is shown in Figure 2 As shown. Figure 2As shown, one of the heavy chains of the anti-human PD1 antibody is covalently linked to the p35 subunit of the IL-12 mutant protein through a linker; the p40 subunit of IL-12 is bound to p35 through a disulfide bond between C74 of p35 and C177 of p40. In addition, in order to promote heterodimerization between 1) the antibody heavy chain covalently linked to p35 and 2) the antibody heavy chain not linked to p35, each heavy chain has a mutation in the Fc region to form a knob or hole structure. Figure 2 As shown, the antibody heavy chain covalently linked to p35 has a mutation to form a knob, and the heavy chain not linked to p35 has a mutation to form a hole. In addition, both heavy chains have mutations in the Fc domain, rendering the Fc effector ineffective. The antibody is of IgG1 subclass.

[0413] Fusion proteins were prepared using an inert Fc domain to eliminate potential Fc-mediated depletion and / or Fc receptor binding and further target IL-12 binding and activity to PD1-positive cells.

[0414] Overall, the anti-PD1 portion of the IL-12H10 mutant protein / anti-PD1 fusion protein helps to "anchor" the fusion protein to PD1-positive and IL-12 receptor-positive cells; in this way, the anti-PD1 portion of the fusion protein helps to target IL12 activity to cells that are dual-positive for PD1 and IL-12 receptors.

[0415] The activity of these fusion proteins was evaluated using human primary cells from healthy peripheral blood mononuclear cell (PBMC) donors. Purified CD4 T cells were activated in vitro and then stimulated with different IL-12H10 mutein / anti-PD1 fusion proteins to assess the phosphorylation level of STAT4, which is a readout of IL-12 activity. (Activated CD4 T cells increase the levels of IL-12 receptor and PD1.)

[0416] The half maximal effective concentration (EC 50 ) are shown in Table 13. Each data column is data from an individual PBMC donor.

[0417] Table 13

[0418]

[0419] As shown in Table 13, the EC of H10 / anti-PD1 fusion molecule 50 It was lower than H10 / isotype IgG, indicating that H10 / anti-PD1 fusion molecule had targeting activity on PD1-positive cells.

[0420] The amino acid sequence of the polypeptide of the H10 / TPP-76868 fusion protein is provided below in Table 14. This fusion is also referred to herein as the "H10868" fusion.

[0421] Table 14

[0422]

[0423]

[0424] The amino acid sequence of the H10868 fusion shown in Table 14 includes the following annotated features. In the heavy chain TPP-76868 sequence (SEQ ID NO: 15), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). In the heavy chain TPP-76868 fused to p35 of the H10 sequence (SEQ ID NO:26), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), mutations forming the "knob" of the knob-in-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence [SGGGGSGGGGGSGGGG (SEQ ID NO:27)] connecting the heavy chain and p35 of H10.

[0425] IL-12H10 mutant protein / anti-PD1 TPP-77658 fusion protein

[0426] The amino acid sequence of the polypeptide of the H10 / TPP-77658 fusion protein is provided below in Table 15. This fusion is also referred to herein as the "H10658" fusion.

[0427] Table 15

[0428]

[0429]

[0430] The amino acid sequence of the H10658 fusion shown in Table 15 includes the following annotated features. In the heavy chain TPP-77658 sequence (SEQ ID NO: 5), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), and mutations that form the "hole" of the knob-hole structure: S354C, T366S, L368A and Y407V (EU numbering; underlined). In the heavy chain TPP-77658 fused to p35 of the H10 sequence (SEQ ID NO:25), there are effector null mutations in Fc: L234A, L235A and G237A (EU numbering; underlined), mutations forming the "knob" of the knob-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence [SGGGGSGGGGGSGGGG (SEQ ID NO:27)] connecting the heavy chain and p35 of H10.

[0431] Example 4: Mouse replacement IL-12 variant / anti-PD1 fusion molecule

[0432] This example describes the generation of mouse surrogate IL-12 variants / anti-PD1 molecules and related experiments. IL-12 biology is largely conserved in humans and mice. However, human IL-12 does not cross-react with the mouse IL-12 receptor and therefore cannot be used in preclinical mouse models. Therefore, mouse surrogate IL-12 variants were generated to further evaluate the activity of IL-12 variants.

[0433] Two different mouse alternative IL-12 variants / anti-PD1 molecules were developed. Both alternative IL-12 variants / anti-PD1 molecules contain the same mouse IL-12 mutein, which was selected because it has a similar level of attenuation as the human H10 IL12 mutein (described in Example 1). For ease of production, the mouse IL-12 mutein was prepared as a single polypeptide in which the IL-12 p40 and p35 subunits are covalently linked by a peptide linker (rather than expressing p40 and p35 as separate polypeptide chains). The sequence of the mouse IL12 mutant protein (containing linked p40 and p35 subunits) is: MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFV GGG EKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS GGGGGGS RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKL EF Y PCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA (SEQ ID NO:31). In this sequence, the p40 subunit is the N-terminal portion (i.e., the first in the sequence), the p35 subunit is the C-terminal portion (i.e., the last in the sequence), and the p40 and p35 subunits are separated by a glycine-serine linker sequence (underlined) (GGGGGGS; SEQ ID NO:32). The p40 and p35 portions of the polypeptide contain only the mature portion of the respective polypeptide (i.e., excluding the leader sequence). Compared to wild-type mouse IL-12, the mutant protein has the following mutations (each mutation is underlined in the above sequence; the amino acid numbering is based on the mature polypeptide sequence): p40: GGG (to reduce heparin binding) [mouse p40 residue RIQRKK (amino acid numbering 251-256) is replaced by the sequence GGG]; p35: K34E, H35F and S37P (each mutation is used to attenuate IL-12 activity).

[0434] The two different mouse alternative IL-12 variant / anti-PD1 fusion proteins contain the above-mentioned mouse IL-12 mutant protein, and a PD1 blocking anti-mouse PD1 antibody or a PD1 non-blocking anti-mouse PD1 antibody. Therefore, the two mouse alternative IL-12 variant / anti-PD1 fusion proteins are: 1) a mouse IL-12 mutant protein linked to an anti-mouse PD1 blocking antibody [this fusion is also referred to as "mIL12 mutant protein-PD1 (B)" or "PD1 (B)-mIL12"] and 2) a mouse IL-12 mutant protein linked to an anti-mouse PD1 non-blocking antibody [this fusion is also referred to as "mIL12 mutant protein-PD1 (NB)" or "PD1 (NB)-mIL12"].

[0435] The activity of two mouse alternative IL-12 mutant proteins / anti-PD1 fusion proteins was compared in ex vivo CT26 tumor stimulation to evaluate whether there were differences in the activity of the molecules caused by different anti-PD1 specificities (blocking vs. non-blocking). CT26 tumors (murine colon carcinoma) were harvested from mice implanted with CT26 cells. Tumors were processed to achieve single-cell suspensions of tumor cells and other cells in the tumor microenvironment and then stimulated with one of four different fusion proteins: 1) mouse wild-type IL-12 linked to a mouse Fc domain ("mIL12 wt-Fc"); 2) mouse IL-12 mutant protein linked to a mouse Fc domain ("mIL12 mutant protein-Fc"); 3) mIL12 mutant protein-PD1 (B); or 4) mIL12 mutant protein-PD1 (NB). After 24 hours, the activity of the different fusion proteins was evaluated by the amount of interferon gamma (IFNg) present in the supernatant 24 hours later.

[0436] The results are shown in Figure 3 .exist Figure 3 In the figure, data points are depicted as follows: 1) mIL12 wt-Fc: solid line, filled squares; 2) mIL12 mutant protein-Fc: dashed line, asterisks; 3) mIL12 mutant protein-PD1 (B): solid line, triangles; or 4) mIL12 mutant protein-PD1 (NB): solid line, open circles. Figure 3 As shown, the activities of mIL12 mutant protein-PD1 (B) and mIL12 mutant protein-PD1 (NB) were similar, indicating that PD1 blockade is not required for the activity of PD1 targeting IL12 mutant protein. Figure 3 It was also shown that mIL12 wt-Fc had greater activity than mIL12 mutein-PD1 (B) and mIL12 mutein-PD1 (NB), consistent with the wild-type IL-12 activity of mIL12 wt-Fc. Figure 3 It was also shown that mIL12 mutein-Fc had lower activity than mIL12 mutein-PD1(B) and mIL12 mutein-PD1(NB), consistent with the lack of targeting of mIL12 mutein-Fc.

[0437] The activity of human IL-12 variant / anti-PD1 fusion proteins containing H10 IL-12 mutant proteins was compared to the activity of mouse surrogate IL-12 mutant proteins / anti-PD1-fusion proteins mIL12 mutant proteins-PD1 (B) in a matched assay using CD4 T cells obtained from healthy human donors or isolated from naive mouse splenocytes. The human IL-12 variant / anti-PD1 fusion protein used in this experiment contained H10 IL-12 mutant proteins (described in Example 1) linked to the non-blocking anti-hPD1 antibody TPP-68807 (described in Example 2; which is a derivative of GBT-PD1-0013 and closely related to TPP-77658). This fusion protein is referred to in this experiment as "hIL12 mutant proteins-hPD1 (NB)".

[0438] CD4 T cells were activated in vitro and then stimulated with different fusion proteins as follows. Human CD4 T cells were stimulated with: 1) human wild-type IL-12 (i.e., not specific for PD1) linked to a human isotype (IgG1) control antibody ("hIL12 wt-isotype"); 2) human H10 IL-12 mutein (described in Example 1) fused to a human isotype (IgG1) control antibody ("hIL12 mutein-isotype"); and 3) hIL12 mutein-hPD1 (NB). Mouse CD4 T cells were stimulated with: 1) mouse wild-type IL-12 (i.e., not specific for PD1) fused to a human isotype (IgG1) control antibody ("mIL12 wt-isotype"); 2) mouse IL-12 mutein corresponding to human H10 fused to a human isotype (IgG1) control antibody ("mIL12 mutein-isotype"); and 3) mIL12 mutein-PD1 (B) (described above). For each assay, pSTAT4 was measured by flow cytometry 60 minutes after stimulation of T cells. This assay incorporates analysis of the estimated number of PD1 receptors per cell, and pSTAT4 readings were limited to cells with similar numbers of PD1 receptors per cell to allow for more accurate comparisons. The results are summarized in Table 16 below.

[0439] Table 16

[0440]

[0441] As shown in Table 16, both human and mouse IL12 mutant proteins have similar levels of activity reduction (reduced by about 23,000 times) relative to the corresponding human or mouse wild-type IL12. In addition, the rescue of IL12 mutant protein activity directed by PD1 (measured as the gain of activity of IL12 mutant protein / anti-PD1 antibody fusion protein relative to IL12 mutant protein / non-specific isotype antibody fusion) is also similar to both human and mouse fusion proteins (increased by about 100 times). Overall, this experiment shows that human hIL12 mutant protein-hPD1 (NB) fusion protein and mouse replacement mIL12 mutant protein-PD1 (B) fusion protein are closely matched in terms of IL12 attenuation and rescue activity by adding anti-PD1 antibodies. In addition, given that the above experiments showed that the activities of mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB) fusion proteins are comparable, it can be concluded that in the corresponding experimental systems (humans and mice), the hIL12 mutant protein-hPD1(NB) fusion protein has similar activities to both the mIL12 mutant protein-PD1(B) fusion protein and the mIL12 mutant protein-PD1(NB) fusion protein.

[0442] Example 5: In vivo efficacy studies

[0443] Efficacy studies were performed in two syngeneic mouse tumor models (MC38R and B16F10). MC38R is a murine colon adenocarcinoma cell line. B16F10 is a murine melanoma cell line. The molecules listed in Table 17 were used in these studies.

[0444] Table 17

[0445] Molecule name describe mIL12 mutant protein-PD1 (B) Mouse IL-12 mutein linked to anti-mouse PD1 blocking antibody mIL12 mutant protein-PD1 (NB) Mouse IL-12 mutein linked to anti-mouse PD1 non-blocking antibody mIL12 mutant protein-isoform Mouse IL-12 mutein linked to human IgG1 isotype (control) antibody mIL12wt-isotype Wild-type mouse IL-12 linked to human IgG1 isotype (control) antibody mIL12wt-PD1(B) Wild-type mouse IL-12 linked to anti-mouse PD1 blocking antibody Isotype Human isotype IgG1 (control) antibody mPD1(B) Anti-mouse PD1 blocking antibody mPD1 F2 Anti-mouse PD1 antibody, clone F2 mPD1 RMP1-14 Anti-mouse PD1 antibody, clone RMP1-14

[0446] MC38R Experiment 1

[0447] Methods: 500,000 MC38R tumor cells were implanted subcutaneously into female C57BL / 6 mice. Ten days later, the mice were randomly divided into groups with an average tumor volume of 44-92 mm 3 Treatment groups. Mice were treated with a single subcutaneous dose of 1) mIL12 mutant protein-PD1 (NB), 2) mIL12 mutant protein-isoform, 3) mouse isoform, or 4) mPD1 (B). On the day after randomization, mIL12 mutant protein-PD1 (NB) and mIL12 mutant protein-isoform were administered at 0.05 mg / kg, 0.17 mg / kg, or 0.5 mg / kg, and mouse isoform and mPD1 (B) were administered at 0.5 mg / kg. Tumor volume and body weight were measured twice a week until the end of the study, when the tumor volume of the first mouse reached 2000m 3 . 10 mice / treatment group.

[0448] Results: In this experiment, a single dose of mIL12 mutant protein-PD1(B) administered subcutaneously at 0.05 mg / kg, 0.17 mg / kg or 0.5 mg / kg induced potent dose-dependent tumor growth inhibition (TGI) (61%, 76% and 92%, respectively), while administration of the same dose levels of mIL12 mutant protein-isoforms did not induce significant TGI (-25%, -13% and 45%, respectively). In addition, administration of plain mPD1(B) antibody (i.e., not fused to IL12 mutant protein) also did not induce significant TGI (12%). The lack of TGI in the mPD1(B) antibody treatment group was expected, as its administration dose was 20-fold lower than the normal administration dose of therapeutic PD1 antagonist antibodies and the administration frequency was low (single dose instead of every 3 days, 3 times or once or twice a week). No significant body weight loss (BWL) was observed at any dose level tested for any of these molecules.

[0449] MC38R Experiment 2

[0450] Methods: 500,000 MC38R tumor cells were implanted subcutaneously into female C57BL / 6 mice. Ten days later, the mice were randomly divided into groups with an average tumor volume of 31-131 mm 3 On the day after randomization, mice were treated with a single subcutaneous dose of 1) mIL12 wt-isoform, 2) mIL12 wt-PD1(B), 3) mIL12 mutant-isoform, 4) mIL12 mutant-PD1(B), 5) mouse isoform, or 6) mPD1(B) at 0.5 mg / kg. Tumor volume and body weight were measured twice weekly until the end of the study, when the first mouse reached a tumor volume of 2000 m 3 . 3-10 mice / treatment group.

[0451] Results: mIL12 mutein-PD1(B), mIL12 wt-isoform, and mIL12 wt-PD1(B) all induced robust and significant TGI relative to isotype-treated animals when administered subcutaneously at a single dose of 0.5 mg / kg: 87% TGI for mIL12 mutein-PD1(B), 96% TGI for mIL12 wt-isoform, and 70% TGI for mIL12 wt-PD1(B). Treatment with mIL12 mutein-isoform did not induce significant TGI (14%). Mice treated with mIL12 mutein-PD1(B) did not exhibit BWL, but mice treated with mIL12 wt-isoform or mIL12 wt-PD1(B) showed significant BWL on day 6 after dosing, with mean BWL of 21% and 19%, respectively.

[0452] These results indicate that mIL12 mutant-PD1(B) can induce robust TGI without BWL; this is in contrast to mIL12wt-isoform and mIL12 wt-PD1(B), which both induce robust TGI but induce significant BWL.

[0453] MC38R Experiment 3

[0454] This experiment used MC38R B2M KO cells. In these tumor cells, the B2M gene is missing, so the tumor cells are unable to load antigens to the major histocompatibility (MHC) I complex. This makes the tumors MHC I low or negative and resistant to CD8 T cell killing. Therefore, these tumor cells are also resistant to PD(L)1 therapy.

[0455] Methods: 500,000 MC38R B2M KO tumor cells were implanted subcutaneously into female C57BL / 6 mice. Seven days later, the mice were randomly divided into groups with an average tumor volume of 52-91 mm 3 Treatment groups. Starting from the day after randomization (d0), mice were treated with 1) mIL12 mutant protein-PD1 (NB), 2) mPD1 F2, 3) mPD1 RMP1-14, or 4) mouse isotype. mIL12 mutant protein-PD1 (NB) was administered subcutaneously once at 0.5 mg / kg, mPD1 F2 was administered intraperitoneally at 10 mg / kg once every 3 days for 3 times (Q3D×3), mPD1 RMP1-14 was administered at 10 mg / kg once a week for twice (QW×2), and mouse isotype was administered at 10 mg / kg QW×2. Tumor volume and body weight were measured twice a week until the end of the study, when the tumor volume of the first mouse reached 2000m 3 . 10 mice / treatment group.

[0456] Results: A single dose of mIL12 mutant protein-PD1 (NB) elicited a robust and significant TGI (75%) relative to the mouse isotype. In contrast, mPD1 F2 and mPD1 RMP1-14 failed to elicit a significant TGI against these tumor cells (-23% and 5%, respectively, relative to the mouse isotype). No BWL was observed in any group.

[0457] These results indicate that mIL12 mutant protein-PD1(NB) effectively induces TGI in a PD(L)1-resistant tumor model.

[0458] B16F10 Experiment 1

[0459] Methods: 500,000 B16F10 tumor cells were implanted subcutaneously into female C57BL / 6 mice. Ten days later, the mice were randomly divided into groups with an average tumor volume of 52-111 mm 3 Treatment groups. On the day after randomization (d0), mice were treated with a single subcutaneous dose of 1) mIL12 mutant protein-PD1 (B), 2) mIL12 mutant protein-PD1 (NB), or 3) mouse isotype. mIL12 mutant protein-PD1 (B) and mIL12 mutant protein-PD1 (NB) were administered subcutaneously once at 0.5 mg / kg and 1.5 mg / kg (different treatment groups), respectively, and the mouse isotype was administered at a dose of 1.5 mg / kg.

[0460] Results: A single dose of mIL12 mutant protein-PD1 (B) induced a strong dose-dependent TGI (0.5 mg / kg: 67% TGI; 1.5 mg / kg: 87% TGI). In addition, a single dose of mIL12 mutant protein-PD1 (NB) also induced a strong dose-dependent TGI (0.5 mg / kg: 76% TGI; 1.5 mg / kg: 78% TGI). BWL was not observed in any group.

[0461] These results indicate that the effectiveness of mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB) molecules in inducing TGI in the B16F10 tumor model. In addition, these results indicate that the effectiveness of mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB) molecules is independent of antagonizing the interaction between PD1 and PDL1.

Claims

1. An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and position D93 of SEQ ID NO: 2 (IL-12p40 subunit).

2. The IL-12 variant according to claim 1, wherein the Y167 substitution is Y167A.

3. The IL-12 variant according to any one of claims 1 to 2, wherein the D93 substitution is D93L.

4. The IL-12 variant according to any one of claims 1 to 3, wherein the Y167 substitution is Y167A and the D93 substitution is D93L.

5. The IL-12 variant according to any one of claims 1 to 4, wherein the p40 subunit further comprises one or more mutations to reduce the binding of IL-12 to heparin.

6. The IL-12 variant according to claim 5, wherein the mutation that reduces the binding of IL-12 to heparin comprises substitutions of K258G, S259G and K260G and deletions of R261, E262, K263 and K264 of SEQ ID NO:

2.

7. An isolated human interleukin 12 (IL-12) variant comprising one or both of the following: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (variant IL-12p35 subunit), and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (variant IL-12p40 subunit).

8. An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at one or more of the following positions: F39 (IL-12p35 subunit) of SEQ ID NO: 1, I52 of SEQ ID NO: 1, Y167 of SEQ ID NO: 1, K85 (IL-12p40 subunit) of SEQ ID NO: 2, and D93 of SEQ ID NO:

2.

9. The IL-12 variant according to claim 8, wherein the F39 substitution is F39R or F39A, the I52 substitution is I52E, I52R or I52H, the Y167 substitution is Y167A, the K85 substitution is K85E, and the D93 substitution is D93L.

10. The IL-12 variant according to any one of claims 8 to 9, wherein the p40 subunit further comprises one or more mutations to reduce the binding of IL-12 to heparin.

11. The IL-12 variant according to any one of claims 1 to 10, wherein the IL-12 variant has reduced activity compared to wild-type human IL-12.

12. An isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 10 or 37, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 11, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:

14.

13. The antibody according to claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO:

8.

14. An isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:5, 51 or 52, and the light chain comprises the amino acid sequence of SEQ ID NO:6, wherein the C-terminal lysine of SEQ ID NO:5, 51 or 52 is optionally present.

15. An isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 19, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 20 or 37, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 22, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:

24.

16. An isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 9, 35 or 36, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 38 or 39, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:

41.

17. The antibody according to any one of claims 12 to 16, wherein the antibody does not block the binding of PDL1 to PD1.

18. An isolated fusion protein comprising the human interleukin 12 (IL-12) variant of any one of claims 1 to 11 linked to an anti-PD1 antibody.

19. The fusion protein according to claim 18, wherein the anti-PD1 antibody is the antibody according to any one of claims 12 to 16.

20. The fusion protein according to any one of claims 18 to 19, wherein the IL-12 variant comprises an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and at position D93 of SEQ ID NO: 2 (IL-12p40 subunit).

21. The fusion protein according to any one of claims 18 to 20, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

22. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, wherein the fusion protein comprises the polypeptides of SEQ ID NOs: 5, 25, 6 and 4.

23. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, wherein the fusion protein comprises the polypeptides of SEQ ID NOs: 15, 26, 16 and 4.

24. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding one or more of the IL-12 variants, anti-PD1 antibodies, fusion proteins or polypeptides thereof of any one of claims 1 to 23.

25. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding a human interleukin 12 (IL-12) variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12p35 subunit) and position D93 of SEQ ID NO: 2 (IL-12p40 subunit), wherein the one or more nucleotide sequences comprise the nucleotide sequence of SEQ ID NO: 44 and the nucleotide sequence of SEQ ID NO:

45.

26. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding VH, VL, or both of an antibody that binds to PD1, wherein the polynucleotide comprises the VH nucleic acid sequence of SEQ ID NO: 46, the VL nucleic acid sequence of SEQ ID NO: 47, or both the VH nucleic acid sequence of SEQ ID NO: 46 and the VL nucleic acid sequence of SEQ ID NO:

47.

27. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding any one or more of the heavy chain, light chain, IL-12p40 subunit, or heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD1 antibody, wherein the polynucleotide comprises the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12p40 subunit nucleic acid sequence of SEQ ID NO: 45, the heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49, or each of the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12p40 subunit nucleic acid sequence of SEQ ID NO: 45, and the heavy chain-IL12p35 fusion polypeptide nucleic acid sequence of SEQ ID NO:

49.

28. One or more isolated polynucleotides comprising one or more nucleotide sequences encoding any one or more of the heavy chain, light chain, IL-12p40 subunit, or heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD-1 antibody, wherein the polynucleotide comprises a nucleic acid sequence encoding a heavy chain as an insert of a plasmid deposited with ATCC with ATCC Accession No. PTA-127517, a nucleic acid sequence encoding a light chain as an insert of a plasmid deposited with ATCC with ATCC Accession No. PTA-127519, a nucleic acid sequence encoding an IL-12p40 subunit as an insert of a plasmid deposited with ATCC with ATCC Accession No. PTA-127520, a nucleic acid sequence encoding a heavy chain-IL12 p35 fusion polypeptide as an insert of a plasmid deposited with ATCC with ATCC Accession No. PTA-127518 p35 fusion polypeptide, or the nucleic acid sequence encoding the heavy chain of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127517, the nucleic acid sequence encoding the light chain of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127519, the nucleic acid sequence encoding the IL-12p40 subunit of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127520, and the nucleic acid sequence encoding the heavy chain-IL12 p35 fusion polypeptide of the insert fragment of the plasmid deposited with ATCC having ATCC Accession No. PTA-127518.

29. A vector comprising one or more polynucleotides of any one of claims 24 to 28.

30. An isolated host cell comprising one or more polynucleotides of any one of claims 24 to 28 or the vector of claim 29.

31. A method for producing an IL-12 variant, an anti-PD1 antibody or a fusion protein, the method comprising culturing the host cell of claim 30 under conditions that result in the production of the IL-12 variant, the anti-PD1 antibody or the fusion protein, and optionally further recovering the IL-12 variant, the anti-PD1 antibody or the fusion protein.

32. A pharmaceutical composition comprising the IL-12 variant according to any one of claims 1 to 23, an anti-PD1 antibody or fusion protein, and a pharmaceutically acceptable carrier.

33. A method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 32 or the IL-12 variant, anti-PD1 antibody or fusion protein of any one of claims 1 to 23.

34. The IL-12 variant, anti-PD1 antibody or fusion protein according to any one of claims 1 to 23 for use as a medicament, optionally for use as a medicament for the treatment of cancer.

35. The IL-12 variant, anti-PD1 antibody, fusion protein or method according to any one of claims 33 to 34, wherein the cancer is bladder cancer, breast cancer, clear cell renal carcinoma, head / neck squamous cell carcinoma [head and neck squamous cell carcinoma (SCCHN)], lung squamous cell carcinoma, lung adenocarcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma (RCC), small cell lung cancer (SCLC), triple-negative breast cancer, urothelial carcinoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or disease (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL), endometrial cancer, B-cell acute lymphoblastic leukemia, colorectal cancer (CRC), glioblastoma, uterine cancer, cervical cancer, penile cancer, gastric cancer (GC), or non-melanoma skin cancer.

36. The IL-12 variant, anti-PD1 antibody, fusion protein or method according to any one of claims 33 to 35, wherein the cancer has been previously treated with a PD(L)1 inhibitor other than the anti-PD1 antibody of any one of claims 12 to 17.

Images