Il-12 variants, Anti-PD-1 antibodies, fusion proteins, and uses thereof

Abstract

To provide improved IL-12 variants and related fusion proteins.SOLUTION: An IL-12 variant is provided. An antibody that specifically binds to PD-1 is also provided. A fusion protein of the IL-12 variant and the anti-PD-1 antibody is also provided. Uses of the IL-12 variants, anti-PD-1 antibodies, and fusion proteins, as well as related compositions and methods, are also provided.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to interleukin 12 (IL-12) variants, and methods of making and using said 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.

Background Art

[0002] Interleukin 12 (IL-12) is a cytokine that has 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 boosting 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 (IFNg) production and the cytolytic capacity of CD8 T cells, CD4 T cells, regulatory T cells, and NK cells.

[0003] Preclinical models have shown that IL-12 promotes anti-tumor immunity by its direct action on T cells and NK cells in the tumor microenvironment (TME), as well as its indirect action on antigen-presenting cells. However, preclinical and clinical trials have also shown that IL-12R agonists can have dose-limiting toxicity. These trials hypothesized that significant toxicity is likely to arise from systemic activity.

[0004] Programmed cell death protein 1 (PD1) is an important cell surface receptor that functions to attenuate T cell activation signals and acts as a checkpoint molecule that restricts anti-tumor immunity. Although some PD1 expression has been observed on various 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 tumor microenvironment (TME) compared to circulating T cell subsets.

[0005] Previous studies have targeted cytokines including IL-15, IL-12, and IFN to PD1-positive cells by fusing anti-PD1 antibodies to cytokine mutant protein variants (partial agonists) (Xu, Y. et al., Cancer Immunol Res, 2021, 9(10):1141 - 1157; Codarri Deak, L. et al., Nature, 2022, 610(7930):161 - 172; Hashimoto, M. et al., Nature, 2022, 610(7930):173 - 181, Garcin, G. et al., Nat Commun, 2014, 5:3016).

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, improved IL-12 variants and related fusion proteins are needed.

Means for Solving the Problems

[0007] The present disclosure provides interleukin 12 (IL-12) variants, as well as methods of making and using said variants. The invention also provides anti-PD1 antibodies, as well as fusion proteins comprising such 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 are capable of binding to PD1 simultaneously with the binding of PD1 to PDL1 (e.g., the anti-PD1 antibody binds to a different position on PD1 to which PDL1 binds). In some embodiments, the IL-12 variant / anti-PD1 fusion proteins provided herein have activity biased towards the tumor microenvironment (TME) rather than systemic activity. In some embodiments, the IL-12 variant / anti-PD1 fusion proteins provided herein have an improved therapeutic index compared to wild-type IL-12 and its fusions.

[0008] The present disclosure further encompasses the preparation and manufacture of compositions comprising the IL-12 variants, antibodies, and fusion proteins of the present disclosure, such as agents for the expression of IL-12 variants, anti-PD1 antibodies, and fusion proteins, and the use of the IL-12 variants, antibodies, and fusion proteins.

[0009] In some embodiments, an isolated human interleukin 12 (IL-12) variant is provided herein, comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12 p35 subunit) and an amino acid substitution at position D93 of SEQ ID NO: 2 (IL-12 p40 subunit).

[0010] In some embodiments, an isolated human interleukin 12 (IL-12) variant is provided herein, comprising an amino acid substitution at position Y167A of SEQ ID NO: 1 (IL-12 p35 subunit) and an amino acid substitution at position D93L of SEQ ID NO: 2 (IL-12 p40 subunit).

[0011] In some embodiments, an isolated human interleukin 12 (IL-12) variant is provided herein that comprises one or both of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (variant IL-12 p35 subunit); and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (variant IL-12 p40 subunit).

[0012] In some embodiments, an isolated human interleukin 12 (IL-12) variant is provided herein that comprises an amino acid substitution at one or more positions of F39 (IL-12 p35 subunit) of SEQ ID NO: 1, I52 of SEQ ID NO: 1, Y167 of SEQ ID NO: 1, K85 (IL-12 p40 subunit) of SEQ ID NO: 2, and D93 of SEQ ID NO: 2.

[0013] In some embodiments, an isolated antibody that binds to PD1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL) is provided herein, 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.

[0014] In some embodiments, an isolated antibody that binds to PD1 and comprises a VH amino acid sequence that includes 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 is provided herein.

[0015] In some embodiments, an isolated antibody is provided herein that comprises 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 optional.

[0016] 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.

[0017] In some embodiments, provided herein is an isolated antibody that binds to PD1 and comprises 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.

[0018] 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 optional.

[0019] 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.

[0020] In some embodiments, provided herein is an isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, the fusion protein comprising the polypeptides of SEQ ID NO: 5, 25, 6, and 4.

[0021] In some embodiments, provided herein is an isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, the fusion protein comprising the polypeptides of SEQ ID NO: 15, 26, 16, and 4.

[0022] In some embodiments, provided herein is an isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding a human interleukin 12 (IL-12) variant comprising amino acid substitutions at position Y167 of SEQ ID NO: 1 (IL-12 p35 subunit) and position D93 of SEQ ID NO: 2 (IL-12 p40 subunit), the one or more nucleotide sequences comprising the nucleotide sequence of SEQ ID NO: 44 and the nucleotide sequence of SEQ ID NO: 45.

[0023] In some embodiments, provided herein is an isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding a VH, a VL, or both of an antibody that binds to PD1, the polynucleotide comprising 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.

[0024] In some embodiments, an isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any one or more of a heavy chain, a light chain, an IL-12 p40 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 the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12 p40 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 the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12 p40 subunit nucleic acid sequence of SEQ ID NO: 45, and the heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49, respectively, are provided herein.

[0025] In some embodiments, an isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any one or more of a heavy chain, a light chain, an IL-12 p40 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 encodes the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127517 for the heavy chain, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127519 for the light chain, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127520 for the IL-12 p40 subunit, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127518 for the heavy chain-IL12 p35 fusion polypeptide, or the polynucleotide encodes the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127517 for the heavy chain, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127519 for the light chain, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127520 for the IL-12 p40 subunit, and the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the ATCC accession number PTA-127518 for the heavy chain-IL12 p35 fusion polypeptide, respectively, is provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0027] The present invention can be more easily understood by referring to the following detailed description of the embodiments of the present invention and the examples included herein. It should be understood that the present invention is not limited to a specific production method and can of course be changed. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting.

[0028] Exemplary embodiments (E) of the present invention provided herein include the following.

[0029] An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12 p35 subunit) and an amino acid substitution at position D93 of SEQ ID NO: 2 (IL-12 p40 subunit).

[0030] The IL-12 variant according to E1, wherein the Y167 substitution is Y167A.

[0031] The IL-12 variant according to any one of E1 to E2, wherein the D93 substitution is D93L.

[0032] The IL-12 variant according to any one of E1 to E3, wherein the Y167 substitution is Y167A and the D93 substitution is D93L.

[0033] The IL-12 variant according to any one of E1 to E4, wherein the p40 subunit further comprises one or more mutations that reduce the binding of IL-12 to heparin.

[0034] The IL-12 variant according to E5, wherein the mutations that reduce the binding of IL-12 to heparin include the substitutions of K258G, S259G, and K260G of SEQ ID NO: 2, and the deletions of R261, E262, K263, and K264.

[0035] An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution of Y167A of SEQ ID NO: 1 (IL-12 p35 subunit) and an amino acid substitution of D93L of SEQ ID NO: 2 (IL-12 p40 subunit).

[0036] The IL-12 variant according to E7, wherein the p40 subunit further comprises one or more mutations that reduce the binding of IL-12 to heparin.

[0037] An IL-12 variant described in E8, wherein the mutation that reduces the binding of IL-12 to heparin includes the substitutions of K258G, S259G, and K260G of SEQ ID NO: 2, and the deletions of R261, E262, K263, and K264.

[0038] 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-12 p35 subunit) and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (variant IL-12 p40 subunit).

[0039] E11. An IL-12 variant described in 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.

[0040] E12. An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at one or more positions of F39 (IL-12 p35 subunit) of SEQ ID NO: 1, I52 of SEQ ID NO: 1, Y167 of SEQ ID NO: 1, K85 (IL-12 p40 subunit) of SEQ ID NO: 2, and D93 of SEQ ID NO: 2.

[0041] E13. An IL-12 variant described in E12, wherein the F39 substitution is F39R or F39A.

[0042] E14. An IL-12 variant described in any one of E12 to E13, wherein the I52 substitution is I52E, I52R, or I52H.

[0043] E15. An IL-12 variant described in any one of E12 to E14, wherein the Y167 substitution is Y167A.

[0044] E16. An IL-12 variant described in any one of E12 to E15, wherein the K85 substitution is K85E.

[0045] An IL-12 variant according to any one of E12 to E16, wherein the E17.D93 substitution is D93L.

[0046] An IL-12 variant according to any one of E12 to E17, wherein the E18.p40 subunit further comprises one or more mutations that reduce the binding of IL-12 to heparin.

[0047] An IL-12 variant according to E18, wherein the mutation that reduces the binding of IL-12 to heparin comprises the substitutions K258G, S259G, and K260G of SEQ ID NO: 2, and the deletions of R261, E262, K263, and K264.

[0048] An IL-12 variant according to any one of E1 to E19, wherein the IL-12 variant has one or both of i) a decrease in binding to the human IL-12 receptor as compared to the binding of wild-type human IL-12 to the human IL-12 receptor and ii) a decrease in activity as compared to the activity of wild-type human IL-12 against the human IL-12 receptor.

[0049] 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.

[0050] The antibody according to E21, wherein VH comprises the amino acid sequence of SEQ ID NO: 7 or a variant of SEQ ID NO: 7 that contains 1 to 4 amino acid substitutions at residues other than within the CDRs, and VL comprises the amino acid sequence of SEQ ID NO: 8 or a variant of SEQ ID NO: 8 that contains 1 to 4 amino acid substitutions at residues other than within the CDRs.

[0051] The antibody according to E22, wherein E23.VH contains the amino acid sequence of SEQ ID NO: 7 and VL contains the amino acid sequence of SEQ ID NO: 8.

[0052] E24. An isolated antibody that binds to PD1 and contains a VH amino acid sequence including 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.

[0053] E25. An isolated antibody containing a heavy chain containing the amino acid sequence of SEQ ID NO: 5, 51 or 52 and a light chain containing the amino acid sequence of SEQ ID NO: 6, wherein the C-terminal lysine of SEQ ID NO: 5, 51, or 52 is optional.

[0054] E26. An isolated antibody that binds to PD1 and contains a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH CDR1 contains the amino acid sequence of SEQ ID NO: 19, 35, or 36, VH CDR2 contains the amino acid sequence of SEQ ID NO: 20 or 37, VH CDR3 contains the amino acid sequence of SEQ ID NO: 21, VL CDR1 contains the amino acid sequence of SEQ ID NO: 22, VL CDR2 contains the amino acid sequence of SEQ ID NO: 23, and VL CDR3 contains the amino acid sequence of SEQ ID NO: 24.

[0055] E27. The antibody according to E26, wherein VH contains the amino acid sequence of SEQ ID NO: 17 or a variant of SEQ ID NO: 17 containing 1 to 4 amino acid substitutions in residues other than within the CDR, and VL contains the amino acid sequence of SEQ ID NO: 18 or a variant of SEQ ID NO: 18 containing 1 to 4 amino acid substitutions in residues other than within the CDR.

[0056] E28. The antibody according to E27, wherein VH contains the amino acid sequence of SEQ ID NO: 17 and VL contains the amino acid sequence of SEQ ID NO: 18.

[0057] An isolated antibody that binds to PD1 and comprises a VH amino acid sequence containing the VH CDR1, VH CDR2, and VH CDR3 of the amino acid sequence of SEQ ID NO: 17 and the VL CDR1, VL CDR2, and VL CDR3 of the amino acid sequence of SEQ ID NO: 18.

[0058] An isolated antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 15, 53 or 54 and a light chain containing the amino acid sequence of SEQ ID NO: 16, wherein the C-terminal lysine of SEQ ID NO: 15, 53, or 54 is optional.

[0059] 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 contains the amino acid sequence of SEQ ID NO: 9, 35, or 36, VH CDR2 contains the amino acid sequence of SEQ ID NO: 38 or 39, VH CDR3 contains the amino acid sequence of SEQ ID NO: 21, VL CDR1 contains the amino acid sequence of SEQ ID NO: 12, VL CDR2 contains the amino acid sequence of SEQ ID NO: 40, and VL CDR3 contains the amino acid sequence of SEQ ID NO: 41.

[0060] The antibody according to E31, wherein VH contains the amino acid sequence of SEQ ID NO: 33 and VL contains the amino acid sequence of SEQ ID NO: 34.

[0061] The antibody according to any one of E21 to E32, which does not block the binding of PDL1 to PD1.

[0062] The antibody according to any one of E21 to E33, which does not block the binding of an anti-PD1 antibody that inhibits the interaction between PD1 and PDL1 to PD1.

[0063] The antibody according to any one of E21 to E34, which has a modification in the Fc domain that reduces binding to the Fc gamma receptor.

[0064] An isolated fusion protein comprising a human interleukin 12 (IL-12) variant as described in any one of E1 to E20 linked to an anti-PD1 antibody.

[0065] The fusion protein according to E36, wherein the anti-PD1 antibody is an antibody as described in any one of E21 to E34.

[0066] The fusion protein according to 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-12 p35 subunit) and an amino acid substitution at position D93 of SEQ ID NO: 2 (IL-12 p40 subunit).

[0067] The fusion protein according to E38, wherein the Y167 substitution is Y167A and the D93 substitution is D93L.

[0068] The fusion protein according to 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-12 p35 subunit) and ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (IL-12 p40 subunit).

[0069] The fusion protein according to 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.

[0070] The fusion protein according to any one of E36 to E41, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8.

[0071] The fusion protein according to any one of E36 to E41, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), 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.

[0072] E44. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, the fusion protein comprising the polypeptides of SEQ ID NOs: 5, 25, 6, and 4.

[0073] E45. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, the fusion protein comprising the polypeptides of SEQ ID NOs: 15, 26, 16, and 4.

[0074] E46. An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding one or more of the IL-12 variants, anti-PD1 antibodies, fusion proteins or polypeptides thereof according to any one of E1 to E45.

[0075] E47. One or more nucleotide sequences encoding a human interleukin 12 (IL-12) variant comprising amino acid substitutions at positions Y167 of SEQ ID NO: 1 (IL-12 p35 subunit) and D93 of SEQ ID NO: 2 (IL-12 p40 subunit), the one or more nucleotide sequences comprising the nucleotide sequence of SEQ ID NO: 44 and the nucleotide sequence of SEQ ID NO: 45, an isolated polynucleotide or polynucleotides.

[0076] E48. An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding VH, VL, or both of an antibody that binds to PD1, the polynucleotide comprising 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.

[0077] An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any one or more of the heavy chain, light chain, IL-12 p40 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-12 p40 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 the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12 p40 subunit nucleic acid sequence of SEQ ID NO: 45, and the heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO: 49.

[0078] An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any one or more of a heavy chain, a light chain, an IL-12 p40 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 heavy chain encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127517, the light chain encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127519, the IL-12 p40 subunit encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127520, the heavy chain-IL12 p35 fusion polypeptide encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127518, or the heavy chain encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127517, the light chain encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127519, the IL-12 p40 subunit encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127520, and the heavy chain-IL12 p35 fusion polypeptide encodes a nucleic acid sequence of an insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127518, respectively, a polynucleotide.

[0079] E51. The polynucleotide or polynucleotides according to any one of E46 to E50, wherein the polynucleotide is RNA or DNA.

[0080] E52. The polynucleotide or polynucleotides according to any one of E46 to E51, wherein the polynucleotide comprises at least one chemical modification.

[0081] The polynucleotide or polynucleotides according to E52, wherein the chemical modification is selected from pseudouridine, 1-methylpseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0082] The polynucleotide or polynucleotides according to any one of E46 to E53, wherein the polynucleotide does not contain a chemical modification.

[0083] A vector comprising the polynucleotide or polynucleotides according to any one of E46 to E54.

[0084] An isolated host cell comprising the polynucleotide or polynucleotides according to any one of E46 to E54 or the vector according to E55.

[0085] A method for producing an IL-12 variant, an anti-PD1 antibody, or a fusion protein, comprising culturing the host cell according to E56 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.

[0086] A pharmaceutical composition comprising an IL-12 variant, an anti-PD1 antibody, or a fusion protein according to any one of E1 to E45 and a pharmaceutically acceptable carrier.

[0087] A method for treating cancer in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to E58 or an IL-12 variant, anti-PD1 antibody, or fusion protein according to any one of E1 to E45.

[0088] For use as a medicament, optionally for use as a medicament for cancer, an IL-12 variant, anti-PD1 antibody, or fusion protein according to any one of E1 to E45.

[0089] For use in the treatment of cancer, an IL-12 variant, anti-PD1 antibody, or fusion protein according to any one of E1 to E45.

[0090] E62. The cancer is bladder cancer, breast cancer, clear cell renal carcinoma, head and neck squamous cell carcinoma [squamous cell carcinoma of the head and neck (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 cancer, 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 lymphoblastic leukemia, colorectal cancer (CRC), glioblastoma, uterine cancer, cervical cancer, penile cancer, gastric cancer (GC), non-melanoma skin cancer, NSCLC previously treated with a platinum-based treatment and / or a checkpoint inhibitor (e.g., a PD(L1) 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, an IL-12 variant, an anti-PD1 antibody, a fusion protein, or a method according to any of E59 to E61.

[0091] E63. The cancer is an IL-12 variant, an anti-PD1 antibody, a fusion protein, or a method according to any of E59 to E62, which has been previously treated with a PD(L)1 inhibitor different from the anti-PD1 antibody according to any one of E21 to E35.

[0092] E64. The cancer is an IL-12 variant, an anti-PD1 antibody, a fusion protein, or a method according to any of E59 to E62, which has been treated in combination with a PD(L)1 inhibitor different from the anti-PD1 antibody according to any one of E21 to E35.

[0093] The section headings used in this specification are for purposes of organization only and are not to be construed as limiting the subject matter described.

[0094] All references cited in this specification, including patent applications, patent publications, and UniProtKB accession numbers, are hereby incorporated by reference as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

[0095] For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al. (eds.) (2003)); METHODS In the ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor (eds.) (1995)), Harlow and Lane (eds.) (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney (ed.) (1987)); Oligonucleotide Synthesis (M.J. Gait (ed.), 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis (ed.), 1998) Academic Press; Animal Cell Culture (R.I. Freshney (ed.), 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell (eds.), 1993 - 8) J.Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwerr (eds)); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos (eds.), 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (eds.), 1994); Current Protocols in Immunology (J.E. Coligan et al. (eds.), 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty (ed.), IRL Press, 1988 - 1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean (eds.), Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow And D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and J.D. Capra (eds.), Harwood Academic Publishers, 1995); and other widely used methods described in or referenced in its updated versions, the techniques and procedures described or referenced herein, such as those, are generally well understood and commonly used by those skilled in the art using conventional methods.

[0096] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art.

[0097] As used herein, the singular forms "a", "an" and "the" include plural referents unless otherwise indicated. For example, "an" antibody includes one or more antibodies.

[0098] When aspects or embodiments of the present invention are described from the perspective of a Markush group of alternatives or other classifications, the present invention includes not only the entire recited group as a whole, but also each member of the group individually, and all possible subgroups of the main group, as well as the main group lacking one or more of the group members. The present invention also contemplates specifically excluding any one or more of the group members in the claimed invention.

[0099] Any example following the terms "e.g." or "for example" is not meant to be exhaustive or limiting.

[0100] 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 vary by up to 10% below or above the recited numerical 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.

[0101] "Antibody" refers to an immunoglobulin molecule that can specifically bind to a target, e.g., a polypeptide, carbohydrate, polynucleotide, lipid, etc., by at least one antigen recognition site located within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" may include any type of antibody (e.g., monospecific, bispecific), a portion of an intact antibody that retains the ability to bind to a given antigen (e.g., an "antigen-binding fragment"), and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site.

[0102] Antibodies include antibodies of any class, e.g., IgG, IgA, or IgM (or subclasses thereof), etc., and the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes according to the amino acid sequence of the constant region of their heavy chain (HC). There are five major classes of immunoglobulins, namely, IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0103] Examples of antibody-antigen binding fragments and modified constructs include: (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CH1 domains); (ii) F(ab’)2 fragments (divalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region); and (iii) Fv fragments consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that enables the VL and VH regions to pair and form a monovalent molecule (known as a 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 included.

[0104] Furthermore, antibodies are further included in which the C-terminal lysine (K) amino acid residue on the heavy chain polypeptide is missing (for example, the human IgG1 heavy chain contains a terminal lysine). As is known in the art, the C-terminal lysine is cleaved during antibody production, resulting in an antibody having a heavy chain lacking the C-terminal lysine. Alternatively, the antibody heavy chain can be produced using a nucleic acid that does not contain the C-terminal lysine.

[0105] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FR) connected by three complementarity-determining regions (CDR), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of the antibody. In particular, when a variant of a subject variable region having a substitution among the amino acid residues outside the CDR regions (i.e., within the framework regions) is desired, appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies containing the CDR1 and CDR2 sequences in the same canonical class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987).

[0106] In certain embodiments, the identification of the residues comprising the CDRs and the binding site of the antibody is achieved by elucidating the structure of the antibody or the structure of the antibody-ligand complex. In certain embodiments, it can be achieved by any of a variety of techniques known to those of skill in the art, such as X-ray crystallography. In certain embodiments, a variety of analytical methods can be used to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, the extended definition, and the conformation definition.

[0107] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214 - 218. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901 - 917; Chothia et al., 1989, Nature, 342:877 - 883. The extended definition is a combination of the Kabat definition and the Chothia definition. The AbM definition uses an integrated set of computer programs created by the Oxford Molecular Group for modeling antibody structures. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268 - 9272; "AbM (trademark), A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from the primary sequence using a combination of knowledge databases such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3:194 - 198 and ab initio methods. The contact definition is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732 - 745. In another approach referred to herein as the "conformation definition" of CDR, the position of the CDR can be identified as residues that make an enthalpic contribution to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156 - 1166.Still other CDR boundary definitions do not strictly follow one of the above methods, but nevertheless, these can be shorter or longer based on predictions or experimental findings that certain residues or groups of residues do not significantly affect antigen binding and will overlap at least in part with the Kabat CDRs. As used herein, CDR may refer to CDRs defined by any method known in the art, including combinations 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, the CDRs can be defined according to any one or more of the Kabat, Chothia, extended, AbM, contact, or conformation definitions.

[0108] The "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. The IgG heavy chain constant region contains three contiguous immunoglobulin domains (CH1, CH2, and CH3), along with the hinge region between the CH1 and CH2 domains. The IgG light chain constant region contains a single immunoglobulin domain (CL).

[0109] "Fc domain" refers to the portion of an immunoglobulin (Ig) molecule that correlates with the crystallizable fragment obtained by papain digestion of the Ig molecule. As used herein, the term refers to the two-chain constant region of an antibody, where each chain does not include the first constant region immunoglobulin domain. Within the Fc domain, there are two "Fc chains" (e.g., "first Fc chain" and "second Fc chain"). "Fc chain" generally refers to the C-terminal portion of the antibody heavy chain. Thus, an 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 N-terminus of these domains.

[0110] The boundaries of the Fc chain may vary, but the human IgG heavy chain Fc chain is typically defined as including residue C226 or P230 relative to its carboxyl terminus, and the numbering is according to the EU index as described by Edelman et al., Proc. Natl. Acad. Sci. USA 1969;63(1):78-85, and Kabat et al., 1991. Typically, the Fc chain includes from about amino acid residue 236 to about 447 of the human IgG1 heavy chain constant region. "Fc chain" may refer to this polypeptide alone or in the context of a larger molecule (e.g., in an antibody heavy chain or an Fc fusion protein).

[0111] A "functional" Fc domain refers to an Fc domain having 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. Such effector functions generally require that the Fc domain be associated with a binding domain (e.g., an antibody variable region), and can be evaluated using various assays known in the art for assessing such antibody effector functions.

[0112] A "native sequence" Fc chain refers to an Fc chain that includes an amino acid sequence identical to that of an Fc chain found in nature. A "variant" Fc chain includes an amino acid sequence that differs from that of the native sequence Fc chain by at least one amino acid modification.

[0113] A "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. Further, in contrast to polyclonal antibody formulations, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed to require the production of antibodies by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. In another example, monoclonal antibodies can be isolated from phage libraries such as those generated using the techniques described by McCafferty et al., 1990, Nature, 348:552-554.

[0114] A "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or having an amino acid sequence made using any of the techniques for making a fully human antibody. For example, a fully human antibody can be obtained by using a commercially available mouse engineered to express a particular human immunoglobulin protein or by phage, yeast, or ribosome display techniques for preparing a fully human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0115] A "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0116] A "humanized" antibody refers to a chimeric antibody containing minimal sequences derived from non-human immunoglobulins, which is a non-human (e.g., murine) antibody. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues derived from the CDRs of the recipient have been replaced with residues derived from the CDRs of a non-human species (donor antibody), such as a mouse, rat, or rabbit, that have the desired specificity, affinity, and capacity. A humanized antibody may contain residues not found in the recipient antibody, the transferred CDRs, or the framework sequences, but which are included to further refine and optimize antibody performance.

[0117] "Antigen" refers to a molecular entity used for immunization of an immunocompetent vertebrate to produce antibodies that recognize the antigen or for screening an expression library (e.g., particularly, a phage, yeast, or ribosome display library) for antibody selection. As used herein, antigen is more broadly referred to and is generally intended to include a target molecule specifically recognized by an antibody, and thus includes fragments or mimetics of molecules used in an immunization process to generate an antibody or in library screening to select an antibody.

[0118] "Epitope" refers to the site or region of an antigen to which an antibody specifically binds, when determined by any method well known in the art, e.g., a site or region containing residues that interact with the antibody. For example, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999, there are many methods well known in the art for mapping and characterizing the location of epitopes on a protein, including elucidation of the crystal structure of the antibody-antigen complex, competitive assays, gene fragment expression assays, epitope mapping, and synthetic peptide-based assays. Further, or alternatively, during the discovery process, information about a desired epitope can be elucidated by generation and characterization of antibodies. From this information, it is then possible to competitively screen antibodies for binding to the same epitope.

[0119] Furthermore, the epitope to which an antibody binds can be determined by systematic screening using overlapping peptides derived from the antigen and determining binding by the antibody. According to a gene fragment expression assay, the open reading frame encoding the antigen is fragmented randomly or by a specific gene construct, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. The gene fragments can be produced by PCR in vitro, for example, in the presence of radiolabeled amino acids, and then transcribed and translated into protein. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0120] 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 further examples, mutagenesis of the antigen, domain exchange experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, or necessary for epitope binding.

[0121] At its most detailed level, an epitope for the interaction of an antigen and an antibody can be defined by the spatial coordinates that define the atomic contacts present during the antigen-antibody interaction, as well as information regarding its relative contribution to the binding thermodynamics. At a less detailed level, an epitope can be characterized by the spatial coordinates that define the atomic contacts between the antigen and the antibody. At an even less detailed level, an epitope can be characterized by the amino acid residues it contains, defined by specific criteria, for example, by the distance between atoms (e.g., heavy atoms, i.e., non-hydrogen atoms) in the antibody and the antigen. At an even less detailed level, an epitope can be characterized by function, for example, by competitive binding with other antibodies. Also, an epitope can be more generally defined as including amino acid residues whose substitution by another amino acid alters the characteristics of the interaction between the antibody and the antigen (e.g., using alanine scanning).

[0122] From the fact that descriptions and definitions of epitopes are obtained at different levels of detail depending on the epitope mapping method used, it follows that comparisons of epitopes for different antibodies on the same antigen can be made at different levels of detail as well.

[0123] For example, epitopes described at the amino acid level, as 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 set of amino acid residues. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be distinct (unique) if no amino acid residues are shared by the epitopes.

[0124] Yet another method that can be used to characterize an antibody is to use a competitive assay with other antibodies known to bind the same antigen to determine whether the antibody of interest binds to the same epitope as the other antibodies. Competitive assays are well known to those skilled in the art. Epitopes characterized by competitive binding are said to overlap if the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody precludes the simultaneous or sequential binding of the other antibody. Epitopes are said to be distinct (unique) if the antigen can simultaneously accommodate the binding of both corresponding antibodies.

[0125] Epitopes can be linear or conformational. In a linear epitope, all points of interaction between the protein and an interacting molecule (such as an antibody) lie linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" contains non-contiguous polypeptides (or amino acids) within the antigen protein to which an antibody specific for the epitope binds.

[0126] The term "binding affinity" refers to the total 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, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed as the dissociation constant (K D) can be represented by. Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens faster and tend to remain bound for longer. In particular, the term "binding affinity" is intended to refer to the dissociation rate of a particular antigen-antibody interaction. K D is the "off-rate (k off )" or "k d ", which is also called, the rate of dissociation, the rate of binding, or the ratio to the "on-rate (k on )" or "k a ". Thus, K D is k off / k on (or k d / k a ) and is expressed as molar concentration (M). The smaller K D is, the stronger the binding affinity is. Thus, a K D of 1 μM shows a weaker binding affinity compared to a K D of 1 nM. The K D value of an antibody can be determined using well-established methods in the art. One exemplary method for determining the K D of an antibody is by using surface plasmon resonance (SPR), typically by using a biosensor system such as a BIACORE system. BIACORE kinetic analysis involves analyzing the binding and dissociation of an antigen from a chip on which a molecule (e.g., a molecule containing an epitope-binding domain) is immobilized on the surface. Another method for determining the K D of an antibody is by using biolayer interferometry, typically by using the OCTET® technology (Octet QK e system, ForteBio). Alternatively, or additionally, the KinExA assay (binding equilibrium exclusion method) available from Sapidyne Instruments (Boise, ID) can also be used.

[0127] The term "monospecific antibody" refers to an antibody that contains one or more antigen-binding sites per molecule, such that any and all of the binding sites of the antibody specifically recognize the same epitope on an antigen. Thus, when a monospecific antibody has more than one antigen-binding site, the binding sites compete with each other for binding to one antigen molecule.

[0128] The term "bispecific antibody" refers to a molecule having binding specificities for at least two different epitopes. In some embodiments, the bispecific antibody can bind to two different antigens simultaneously. In other embodiments, the two different epitopes may be present on the same antigen.

[0129] The term "half maximal effect concentration (EC 50 )" refers to the concentration of a therapeutic agent that elicits a response that is half-way between the baseline and the maximum after a specified exposure time. The therapeutic agent may cause inhibition or stimulation. The EC 50 value is commonly used and is used herein as a measure of potency.

[0130] An "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist encompasses substances (such as antibodies) that bind to a molecule and promote the activity of that molecule.

[0131] An "antagonist" refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or effect of another molecule, such as a receptor. The term antagonist encompasses substances (such as antibodies) that bind to a molecule and prevent or reduce the activity of that molecule.

[0132] As used herein with respect to antibodies, the term "competes" means that a first antibody binds to an epitope in a manner sufficiently similar to the binding of a second antibody such that the result of the second antibody's binding to its cognate epitope is detectably reduced in the presence of the first antibody as compared to the binding of the second antibody in the absence of the first antibody. It is also possible, but not necessary, that the binding of the first antibody to its epitope is also detectably reduced in the presence of the second antibody. That is, 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, antibodies are said to "cross-compete" with each other for binding to these respective epitopes if each antibody inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same extent, to a greater extent, or to a lesser extent, and detectably so. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of ordinary skill in the art will understand, based on the teachings provided herein, that such competing or cross-competing antibodies are encompassed and may be useful for the methods disclosed herein.

[0133] A standard competition assay can be used to determine whether two antibodies compete with each other. One suitable assay for antibody competition involves the use of surface plasmon resonance (SPR) technology, typically using a biosensor system (such as a BIACORE system) to measure the degree of interaction, including the use of Biacore technology. For example, SPR can be used in an in vitro competition binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay for measuring antibody competition uses an ELISA-based approach.

[0134] The "Fc receptor" (FcR) refers to a receptor that binds to the Fc domain of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors), and examples include receptors of the FcgRI, FcgRII, and FcgRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Examples of FcgRII receptors include FcgRIIA ("activating receptor") and FcgRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcgRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcgRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu.Rev.Immunol. 1997;15:203-234). FcRs are reviewed, for example, in 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 also encompassed by the term "Fc receptor" as used 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, for example, 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; WO2004 / 92219).

[0135] "Fragments" or "portions" of an antibody or polypeptide can be made by truncation, for example, by removal of one or more amino acids from the amino terminus, carboxy terminus or both termini of the polypeptide. One, two, three, four, five, six, seven, eight, nine, ten, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80 to 100 or more amino acids can be removed from the amino terminus, carboxy terminus or both termini of the polypeptide to produce a fragment or portion. Fragments or portions can be made by one or more deletions of amino acids from the polypeptide. Fragments or portions can be made by one or more deletions of amino acids from the polypeptide as well as removal of one or more amino acids from the amino terminus, carboxy terminus or both termini of the polypeptide.

[0136] "Effector cell" refers to a leukocyte that expresses one or more FcRs and performs an effector function. In certain embodiments, the effector cell expresses at least FcgRIII and performs an ADCC effector function. 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 blood.

[0137] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cell injury in which cytotoxic effector cells, such as NK cells, neutrophils, and macrophages, which have Fc receptors (FcR) on their surface, can be made to specifically bind to target cells carrying an antigen via secreted Ig bound to the FcR on these cytotoxic effector cells, and then kill the target cells using cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcgRIII, while monocytes express FcgRI, FcgRII, and FcgRIII. To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as those described in U.S. Patent Nos. 5,500,362, 5,821,337, or 6,737,056, can be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 1998; 95: 652-656. Further antibodies with an altered Fc domain amino acid sequence and an increased or decreased ADCC activity are described in U.S. Patent Nos. 7,923,538 and 7,994,290.

[0138] The term "altered" FcR binding affinity or ADCC activity refers to an antibody having an enhanced or decreased activity with respect to one or more FcR binding activities or ADCC activities, compared to a parental antibody, when the antibody and the parental antibody differ in at least one structural aspect. An antibody "showing increased binding" to an FcR binds to at least one FcR with a better affinity than the parental antibody. An antibody "showing decreased binding" to an FcR binds to at least one FcR with a lower affinity than the parental antibody. Such an antibody showing decreased binding to an FcR may have little or no detectable binding to the FcR, for example, 0 to 20 percent binding to the FcR, compared to the native sequence IgG Fc domain.

[0139] "Host cell" refers to an individual cell or cell culture that can be, or has been, a recipient of a vector for the incorporation of a polynucleotide insert. Host cells include the progeny of a single host cell, which progeny need not be identical (either in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with the polynucleotides of the present invention.

[0140] "Vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest within a host cell. Examples of vectors include, but are not limited to, plasmids and viral vectors, which may contain naked nucleic acid or nucleic acid associated with delivery aids (e.g., cationic condensing agents, liposomes, etc.). Vectors may contain DNA or RNA. As used herein, "expression vector" refers to a vector containing a gene encoding at least one polypeptide associated with transcription or translation of the gene, and at least one regulatory element (e.g., promoter sequence, poly(A) sequence). Typically, vectors used herein contain a gene encoding at least one antibody, as well as one or more regulatory elements or selectable markers. Vector components may include, for example, one or more of a signal sequence, an origin of replication, one or more marker genes, and suitable transcriptional control elements (such as promoters, enhancers, and terminators). For translation, one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons, can also be included.

[0141] An "isolated" molecule (e.g., an antibody) refers to a molecule that, by virtue of its origin or source of derivation, (1) is not bound to the naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, such as a species, the cells in which it is expressed, a library, etc., (3) is expressed by cells from a different species, or (4) is a molecule that does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell line different from the system in which it naturally terminates will be "isolated" from its naturally associated components. Molecules can also be made substantially free of their naturally associated components by isolation using purification techniques well known in the art.

[0142] A "polypeptide" or "protein" (used interchangeably herein) refers to a chain of amino acids of any length. The chain may be linear or branched. The chain may contain one or more modified amino acids. This term also encompasses amino acid chains that are naturally or modified by any other operation or modification, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation to a labeled component. For example, polypeptides containing one or more analogs of amino acids (including, e.g., non-natural amino acids) and other modifications known in the art are also included within this definition. It is understood that a polypeptide can exist as a single chain or as linked chains.

[0143] "Polynucleotide" or "nucleic acid" (used interchangeably herein) refers to a chain of nucleotides of any length, including DNA and RNA. 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. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications, such as those having uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and those having charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendent moieties, such as those containing proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those having modified linkages (e.g., alpha-anomer nucleic acids, etc.), and unmodified forms of polynucleotides. Further, any of the hydroxyl groups normally present in the sugar can be replaced, for example, with a phosphonate group, phosphorylated, protected with a standard protecting group, or activated to prepare for additional attachment to an additional nucleotide or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized with standard protecting groups.The polynucleotide may also contain analog forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside.

[0144] "Conservative substitution" refers to the replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy biological activity. Structurally similar means that the amino acid has a side chain with a similar length or similar size, such as alanine, glycine and serine. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Specific examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another, or the substitution of one polar residue with another, such as the substitution of arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine, serine with threonine, etc. Specific examples of conservative substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another, the substitution of one polar residue with another, such as the substitution of arginine with lysine, glutamic acid with aspartic acid, or glutamic acid with asparagine, etc. Conservative amino acid substitutions typically include 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.

[0145] The term "identity" or "identical to" refers to the overall relationship between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules or RNA molecules) or between polypeptide molecules. "Identity" measures the percent of identical matches between two or more sequences having a gap alignment handled by a specific mathematical model (e.g., algorithm) of a computer program well known in the art.

[0146] The percent identity of two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., introducing gaps into one or both of the first and second sequences for optimal alignment and ignoring non-identical sequences for comparison purposes). In certain embodiments, the length of the sequences aligned 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 corresponding positions are then compared. If a 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 two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using mathematical algorithms.

[0147] To determine the percent identity, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at the National Center for Biotechnology Information (NCBI). Other alignment programs include the MegAlign® program in the Lasergene® suite of Bioinformatics software (DNASTAR®, Inc., Madison, Wis.). Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), Doolittle (ed.), Academic Press, Inc. Of particular interest are alignment programs that allow gaps in the sequence. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70:173-187 (1997). Sequences can also be aligned using the GAP program that uses the alignment method of Needleman and Wunsch. See J. Mol. Biol. 48:443-453 (1970).

[0148] Also of interest is the BestFit program that uses the Smith and Waterman (1981, Advances in Applied Mathematics 2:482-489) subsequence alignment algorithm to determine sequence identity. The gap creation penalty will generally be in the range of 1-5, usually 2-4, and in some embodiments will be 3. The gap extension penalty will generally be in the range of about 0.01-0.20, and in some examples will be 0.10. The program has default parameters determined by the sequences input for comparison. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package from Madison, WI, USA.

[0149] 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, pages 127-149, 1988, Alan R. Liss, Inc. The percent sequence identity is calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and conjunction penalty: 30.0.

[0150] The terms "increasing", "improving", "decreasing", or "reducing" refer to a value compared to a baseline measurement value, such as a measurement value in the same individual before the start of the treatment described in this specification, or a measurement value in a control individual or subject (or multiple control individuals or subjects) in the absence of the treatment described in this specification. In some embodiments, the "control individual" is an individual suffering from the same form of disease or injury as the individual being treated. In some embodiments, the "control individual" is an individual not suffering from the same form of disease or injury as the individual being treated.

[0151] The term "excipient" refers to any material that, when combined with the active ingredient of interest (e.g., an antibody), enables the active ingredient to retain its biological activity. The choice of excipient will largely depend on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, carriers, diluents, etc. Examples of excipients include one or more of water, saline, phosphate buffered solution, dextrose, glycerol, ethanol, etc., and combinations thereof, and the composition may contain an isotonic agent, such as a sugar, sodium chloride, or a polyalcohol such as mannitol, or sorbitol.

[0152] The terms "treating", "treat", or "treatment" refer to any type of treatment, for example, to relieve, reduce, or delay 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.

[0153] The term "prevent" or "prevention" refers to preventing a disease, condition or disorder in an individual who may have a predisposition to the disease, condition or disorder, but who has not yet experienced or manifested the symptoms of the disease. In some embodiments, prevention is evaluated on a population basis such that a drug is considered to "prevent" a particular disease, disorder or condition if a statistically significant decrease in the incidence, frequency or severity of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition. Prevention can be considered to be achieved when the onset of the disease, disorder or condition is delayed over a predetermined period of time.

[0154] The term "subject", "individual" or "patient" (used interchangeably herein) refers to any animal, including mammals. Mammals according to the present invention include dogs, cats, cows, goats, horses, sheep, pigs, rodents, rabbits, primates, humans, etc., and include mammals in utero. In certain embodiments, humans are the preferred subjects. Human subjects can be of any gender and any stage of development. In some embodiments, the subject is a patient having cancer.

[0155] The term "therapeutically effective amount" refers to the amount of an active ingredient that elicits a biological or medical response in a tissue, system, animal, individual or human as determined by a researcher, veterinarian, physician or other clinician, and includes: (1) preventing a disease; e.g., preventing a disease, condition or disorder in an individual who may have a predisposition to the disease, condition or disorder, but who has not yet experienced or manifested the symptoms of the disease; (2) inhibiting a disease; e.g., inhibiting a disease, condition or disorder in an individual who is experiencing or manifesting the symptoms of the disease, condition or disorder (i.e., stopping or slowing further development of the symptoms); and (3) ameliorating a disease; e.g., ameliorating a disease, condition or disorder in an individual who is experiencing or manifesting the symptoms of the disease, condition or disorder (i.e., reversing the symptoms). may include one or more of.

[0156] IL-12 variant In some embodiments, interleukin 12 (IL-12) variants are provided herein. IL-12 variants are also known as IL-12 "mutant proteins".

[0157] IL-12 is a heterodimer containing two subunits, p35 (also known as IL-12 alpha; it is encoded by the IL-12A gene) and p40 (also known as IL-12 beta; it is encoded by the IL-12B gene). The two IL-12 subunits can form an inter-subunit disulfide bond between C177 of p40 and C74 of p35.

[0158] 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).

[0159] The mature human p35 subunit (SEQ ID NO: 1) is generated from a full-length p35 polypeptide that also includes a 22-amino acid signal peptide that is cleaved during intracellular processing of the initially translated precursor protein. The full-length human p35 amino acid sequence including the signal peptide is available under UniProt accession number P29459 and is provided herein as SEQ ID NO: 28 (the signal peptide is underlined): MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 28).

[0160] All references herein to specific amino acid numbers in the IL-12p35 amino acid sequence are to positions of amino acids in the mature IL-12p35 sequence lacking the signal peptide (not to positions of amino acids in the full-length precursor protein). For example, the amino acid "R1" in the IL-12p35 amino acid sequence refers to the arginine (R) at the 1st position in SEQ ID NO: 1.

[0161] The amino acid sequence of the mature wild-type human IL-12 p40 subunit is provided herein as SEQ ID NO: 2: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 2).

[0162] The mature human p40 subunit (SEQ ID NO: 2) is generated from the full-length p40 polypeptide, which also includes a 22 amino acid signal peptide that is cleaved during the intracellular processing of the initially translated precursor protein. The full-length human p40 amino acid sequence, including the signal peptide, is available under UniProt accession number P29460 and is provided herein as SEQ ID NO: 29 (the signal peptide is underlined): MCHQQLVISWFSLVFLASPLVA IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 29).

[0163] All references herein to specific amino acid numbers in the IL-12p40 amino acid sequence are to positions of amino acids in the mature IL-12p40 sequence lacking the signal peptide (not to positions of amino acids in the precursor full-length protein). For example, amino acid "W2" in the IL-12p40 amino acid sequence refers to tryptophan (W) at the second position in SEQ ID NO: 2.

[0164] As used herein, an IL-12 “variant” or “mutant protein” refers to any IL-12 molecule that contains at least one amino acid change in at least one of the p35 or p40 subunits as 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 as compared to the amino acid sequences of wild-type mature p35 (SEQ ID NO: 1) or p40 (SEQ ID NO: 2).

[0165] In some embodiments, IL-12 variants are provided herein where 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.

[0166] In some embodiments, IL-12 variants are provided herein where 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.

[0167] In some embodiments, an IL-12 variant is provided herein that comprises amino acid substitutions at one or more of the following positions in SEQ ID NO: 1 (p35 subunit): F39, I52, or Y167, and 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.

[0168] In some embodiments, an IL-12 variant is provided herein that has reduced activity and 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, wherein each variant has respective mutations in one or both of the p35 and p40 subunits shown in Table 10.

[0169] In some embodiments, the IL-12 variants provided herein have reduced activity. As used herein, "reduced activity" of an IL-12 variant refers to activity that is reduced as 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 cells in response to IL-12 exposure. STAT4 phosphorylation is an early downstream effect of receptor dimerization induced by IL-12 and thus can serve as a proximal readout for IL-12 activity at the receptor. In another example, IL-12 activity can be evaluated by examining type 1 helper T cell ("Th1")-associated gene transcription, such as IFN gamma gene transcription. pSTAT4 results in upregulation of Th1-associated gene transcription and thus IFN gamma (or other Th1-associated genes) can serve as a downstream readout for IL-12 activity. In another example, IL-12 activity can be indirectly evaluated by measuring the affinity of the IL-12 variant for the IL-12 receptor.

[0170] IL-12 variants having reduced activity can also be described as "less potent" IL-12 variants, "partial agonists", and the like.

[0171] In some embodiments, the IL-12 variants provided herein have reduced binding to the IL-12 receptor as compared to the binding of wild-type IL-12 to the IL-12 receptor. The IL-12 receptor is a heterodimer containing the subunits IL-12R beta1 (see UniProt identification number P42701 for details of human IL-12R beta1) and IL-12R beta2 (see UniProt identification number Q99665 for details of human IL-12R beta2). An IL-12 variant having reduced binding to the IL-12 receptor may be useful, for example, in an environment where the IL-12 variant still binds to the IL-12 receptor with sufficient affinity to activate the receptor in a particular environment, but provides lower activation of the IL-12 receptor as compared to wild-type IL-12. IL-12 activity can be therapeutically useful for activating a subject's immune system, but excessive IL-12 activity can be useful for patients in the event of overstimulation of the immune response and can result in treatment-related adverse events (TRAE) such as cytokine release syndrome (CRS).

[0172] In some embodiments, the IL-12 variants provided herein have an activity 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%, 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.

[0173] In some embodiments, the IL-12 variants provided herein have an activity 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 as compared to the activity of the same amount of the corresponding wild-type IL-12 molecule when tested under the same experimental conditions.

[0174] In some embodiments, the IL-12 variants provided herein have an activity that is reduced to 1 / 2 or less, 1 / 3 or less, 1 / 5 or less, 1 / 10 or less, 1 / 20 or less, 1 / 50 or less, 1 / 100 or less, 1 / 500 or less, 1 / 1000 or less, 1 / 5000 or less, 1 / 10000 or less, 1 / 15000 or less, 1 / 20000 or less, 1 / 23000 or less, 1 / 25000 or less, 1 / 50000 or less, or 1 / 100,000 or less when compared to the activity of the same amount of the corresponding wild-type IL-12 molecule under the same experimental conditions.

[0175] In some embodiments, the affinity of the IL-12 variants provided herein for the IL-12 receptor 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.

[0176] In some embodiments, the affinity of the IL-12 variants provided herein for the IL-12 receptor 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 when compared to the affinity of the corresponding wild-type IL-12 molecule for the IL-12 receptor under the same experimental conditions.

[0177] In some embodiments, the affinity of the IL-12 variants provided herein for the IL-12 receptor is 1 / 2 or less, 1 / 3 or less, 1 / 5 or less, 1 / 10 or less, 1 / 20 or less, 1 / 50 or less, 1 / 100 or less, 1 / 500 or less, 1 / 1000 or less, 1 / 5000 or less, 1 / 10000 or less, 1 / 15000 or less, 1 / 20000 or less, 1 / 23000 or less, 1 / 25000 or less, 1 / 50000 or less, or 1 / 100,000 or less compared to the affinity of the corresponding wild-type IL-12 molecule for the IL-12 receptor when tested under the same experimental conditions.

[0178] Exemplary IL-12 variants provided herein are shown in Example 1 and include those described in the claims and the recited embodiments. The IL-12 variants include, for example, the H10 mutant protein 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.

[0179]

Table 1

[0180] In some embodiments, an IL-12 variant is provided herein, 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 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to 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.

[0181] In some embodiments, 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, and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. IL-12 variants are provided herein. In some embodiments, the K85 substitution is K85E. In some embodiments, the D93 substitution is D93L.

[0182] In some embodiments, 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 at one or more of the following positions in SEQ ID NO: 2 (p40 subunit): K85 or D93, and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. IL-12 variants are provided herein. 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.

[0183] In some embodiments, an IL-12 variant is provided herein, wherein the variant comprises the amino acid substitution Y167 in SEQ ID NO: 1 (p35 subunit) and the amino acid substitution D93L in SEQ ID NO: 2 (p40 subunit), and has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0184] In some embodiments, an IL-12 variant provided herein linked to another protein, such as an antibody, is provided herein. These molecules are referred to as "IL-12 variant fusion proteins" which are described in detail later in this specification.

[0185] Antibody against PD1 The present disclosure also provides an antibody that binds 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 I transmembrane receptor originally identified in a T cell line that undergoes apoptosis induced by activation. PD1 is expressed on various immune cells such as T cells, B cells, and macrophages. Ligands for PD1 are PD-L1 (B7-H1) and PD-L2 (B7-DC), which are B7 family members. PD1 downregulates immune cell activity; thus, 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 from immune cells, and increased anti-tumor responses of immune cells.

[0186] 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 derived from non-human species such as cynomolgus PD1, as well as different forms of PD1. In some embodiments, the antibody may be completely specific for human PD1 and may not exhibit species cross-reactivity (e.g., does not bind to mouse PD1) or other types of cross-reactivity (e.g., does not bind to other receptors of the tumor necrosis factor receptor family). As used herein, the term PD1 refers to naturally occurring human PD1 unless otherwise contextually indicated. Thus, "PD1 antibody", "anti-PD1 antibody" or other similar nomenclature means any antibody (as defined herein), its isoform, fragment or derivative that binds or reacts with PD1.

[0187] There are a number of different anti-PD1 antibodies developed for the treatment of cancer, such as nivolumab and pembrolizumab. Typically, these antibodies produce a therapeutic effect by reducing the biological activity of PD1, such as 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", "inhibiting" or "antagonist" anti-PD1 antibodies.

[0188] 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 at the same time that 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 for their ability to bind to PD1 (even if they do not inhibit PD1 activity mediated by the PD1-PDL1 / PDL2 interaction). For example, non-blocking anti-PD1 antibodies can be used to target a molecule linked to the non-blocking anti-PD1 antibody to PD1-expressing cells such as T cells.

[0189] In some embodiments, the non-blocking anti-PD1 antibodies provided herein can bind to PD1 simultaneously while PD1 is bound by the blocking anti-PD1 antibody.

[0190] As used herein, the term "PD1-binding" antibody refers to both blocking and non-blocking anti-PD1 antibodies.

[0191] In some embodiments, the anti-PD1 antibodies of the present disclosure include antibodies that are either i) competitive for binding to human PD1 or ii) bind to the same epitope as, or both, the amino acid sequence of the heavy chain variable region set forth as SEQ ID NO: 7 and the amino acid sequence of the light chain variable region set forth as SEQ ID NO: 8. In some embodiments, the anti-PD1 antibodies of the present disclosure include antibodies that are either i) competitive for binding to human PD1 or ii) bind to the same epitope as, or both, the amino acid sequence of the heavy chain variable region set forth as SEQ ID NO: 17 and the amino acid sequence of the light chain variable region set forth as SEQ ID NO: 18. In some embodiments, the anti-PD1 antibodies of the present disclosure include antibodies that are either i) competitive for binding to human PD1 or ii) bind to the same epitope as, or both, the amino acid sequence of the heavy chain variable region set forth as SEQ ID NO: 33 and the amino acid sequence of the light chain variable region set forth as SEQ ID NO: 34.

[0192] The anti-PD1 antibodies of the present disclosure 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-chain (ScFv), mutants thereof, fusion proteins containing antibody fragments (e.g., domain antibodies), humanized antibodies, as well as any other modified configurations of immunoglobulin molecules containing antigen-binding sites of the required 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 or humanized antibody. In some embodiments, the anti-PD1 antibody is a chimeric antibody. In some embodiments, the anti-PD1 antibodies provided herein are of the IgG1 subclass. In some embodiments, the anti-PD1 antibodies provided herein have a knob-in-hole mutation in the Fc chain to promote inter-chain heterodimerization. In some embodiments, the anti-PD1 antibodies provided herein have a mutation in the Fc chain to reduce the binding affinity for human Fc gamma receptors.

[0193] In some embodiments, the present invention provides an antibody or a variant thereof having a light chain variable region (VL) sequence and a heavy chain variable region (VH) sequence as found in Table 2. In Table 2, the underlined sequences are CDR sequences (Kabat definition), and the bold sequences are CDR sequences (Chothia definition).

[0194]

Table 2

[0195] In some embodiments, antibodies comprising the VH set forth in SEQ ID NO:7 and the VL set forth in SEQ ID NO:8 are provided herein. In some embodiments, antibodies comprising the VH set forth in SEQ ID NO:17 and the VL set forth in SEQ ID NO:18 are provided herein. In some embodiments, antibodies comprising the VH set forth in SEQ ID NO:33 and the VL set forth in SEQ ID NO:34 are provided herein.

[0196] The present invention also provides CDR portions of antibodies against PD1. Determination of the CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs can be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In another approach referred to herein as "conformationally defined" CDRs, the positions of the CDRs can be identified as residues that make an enthalpic contribution to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Generally, "conformational CDRs" include the Kabat CDRs and residue positions within the framework zones that are constrained to maintain an appropriate loop structure for the antibody to bind to a specific antigen. Determination of conformational CDRs is well within the skill of the art. In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other embodiments, the CDRs are extended, AbM, conformational, or contact CDRs. In other words, in embodiments having more than one CDR, the CDRs can be any of Kabat, Chothia, extended, AbM, conformational, contact CDRs, or combinations thereof.

[0197] In some embodiments, the antibody comprises the three CDRs of the heavy chain variable region shown in Table 2. In some embodiments, the antibody comprises the three CDRs of the light chain variable region shown in Table 2. In some embodiments, the antibody comprises the three CDRs of the heavy chain variable region shown in Table 2 and the three CDRs of the light chain variable region shown in Table 2.

[0198] Table 3 provides examples of the CDR sequences of the anti-PD1 antibodies provided herein. CDRs not shown with any particular CDR definition in Table 3 have the same CDR definition according to Chothia, Kabat, and the extended definition.

[0199] [Table 3]

[0200] In some embodiments, the anti-PD1 antibodies provided herein comprise three light chain CDRs and three heavy chain CDRs derived from the antibodies shown in Table 3.

[0201] In some embodiments, the anti-PD1 antibody comprises either i) a full-length heavy chain with or without a C-terminal lysine, or ii) one or both of the full-length light chains. The amino acid sequences of the full-length heavy and light chains of the exemplary anti-PD1 antibodies provided herein are shown in Table 4 below.

[0202] [Table 4-1]

[0203] [Table 4-2]

[0204] In Table 5, the amino acid sequence of "TPP-77658 heavy chain" (SEQ ID NO: 5) contains the following annotated features in the Fc region: effector null mutations L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined). The amino acid sequence of "TPP-77658 heavy chain without hole mutations" (SEQ ID NO: 51) is the same as SEQ ID NO: 5 except that it does not contain the mutations that form the hole; it still contains the effector null mutations L234A, L235A, and G237A (underlined). The amino acid sequence of "TPP-77658 heavy chain without hole or effector null mutations" (SEQ ID NO: 52) is the same as SEQ ID NO: 5 except that it does not contain the hole or effector null mutations of SEQ ID NO: 5; on the contrary, it contains the corresponding wild-type amino acids.

[0205] In Table 5, the amino acid sequence of "TPP-76868 heavy chain" (SEQ ID NO: 15) contains the following annotated features in the Fc region: effector null mutations L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined). The amino acid sequence of "TPP-76868 heavy chain without hole mutations" (SEQ ID NO: 53) is the same as SEQ ID NO: 15 except that it does not contain the mutations that form the hole; it still contains the effector null mutations L234A, L235A, and G237A (underlined). The amino acid sequence of "TPP-76868 heavy chain without hole or effector null mutations" (SEQ ID NO: 54) is the same as SEQ ID NO: 15 except that it does not contain the hole or effector null mutations of SEQ ID NO: 15; on the contrary, it contains the corresponding wild-type amino acids.

[0206] In Table 5, the amino acid sequence of "TPP-68807 heavy chain" (SEQ ID NO: 42) contains the following annotated features in the Fc: effector null mutations L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined). The amino acid sequence of "TPP-68807 heavy chain without hole mutations" (SEQ ID NO: 55) is the same as SEQ ID NO: 42 except that it does not contain the mutations that form the hole; it still contains the effector null mutations L234A, L235A, and G237A (underlined). The amino acid sequence of "TPP-68807 heavy chain without hole or effector null mutations" (SEQ ID NO: 56) is the same as SEQ ID NO: 5 except that it does not contain the hole or effector null mutations of SEQ ID NO: 42; on the contrary, it contains the corresponding wild-type amino acids.

[0207] In certain embodiments, the antibodies described herein include an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the anti-PD1 antibodies provided herein are IgG1 antibodies.

[0208] 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 these properties, as well as variants with enhanced or decreased activity or affinity. For example, the amino acid sequence can be mutated to obtain an antibody having a desired binding affinity for PD1. Modification of polypeptides is routine in the art and need not be described in detail herein. Examples of modified polypeptides include conservative substitutions of amino acid residues, deletions or additions of one or more amino acids that do not significantly and detrimentally alter the functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides having the use of chemical analogs.

[0209] Modifications or mutations can also be made within the framework or constant regions to increase the half-life of the antibodies provided herein. See, for example, PCT Publication No. WO00 / 09560. Mutations within the framework or constant regions can be made to alter the immunogenicity of the antibody, provide sites for covalent or non-covalent attachment to another molecule, or alter properties such as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. In some embodiments, 1 to 5 or fewer conservative amino acid substitutions are made within the framework or constant regions. In other embodiments, 1 to 3 or fewer conservative amino acid substitutions are made within the framework or constant regions. According to the present invention, a single antibody may have mutations within any one or more of the CDRs or framework regions of the variable domain, or within the constant region.

[0210] In some embodiments, the antibody comprises a modified constant region with increased or decreased binding affinity for human Fc gamma receptors and is immunologically inert or partially inert, e.g., does not induce complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate microglia, or has reduced activity (compared to an unmodified antibody) in any one or more of the following, namely, induction of complement-mediated lysis, stimulation of ADCC, or activation of microglia. Different modifications of the constant region can be used to achieve an 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.

[0211] For example, in some embodiments, the constant region of the antibodies provided herein is modified to have a reduced binding affinity for human Fc gamma receptors. These antibodies are also referred to as "effector null" or having an "inactive Fc domain." 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 (numbering according to EU nomenclature).

[0212] Modifications include glycosylated and non-glycosylated polypeptides, as well as polypeptides having other post-translational modifications such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions within these 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 the protein (Boyd et al., 1996, Mol. Immunol., 32:1311-1318; Wittwe and Howard, 1990, Biochem., 29:4175-4180), as well as the intramolecular interactions between the glycopeptide moiety that can affect conformation and the presented three-dimensional surface of the glycopeptide (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech., 7:409-416). Oligosaccharides can also act to target a given glycopeptide to a particular molecule based on specific recognition structures. Antibody glycosylation has also been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, antibodies produced by CHO cells showing tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisected GlcNAc, have been reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

[0213] In some embodiments, the present disclosure provides an anti-PD1 antibody containing a variable region shown in Table 2, a CDR shown in Table 3, or a mutation in the heavy and light chain sequences shown in Table 4, wherein such variant polypeptide has 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 to any of the amino acid sequences disclosed in Table 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 present disclosure.

[0214] In some embodiments, an anti-PD1 antibody comprising VH and VL is provided herein, wherein the antibody VH has an amino acid sequence encoded by a nucleic acid sequence within the nucleic acid sequence of the insert of the plasmid deposited with the ATCC having ATCC accession number PTA-127517, and the antibody VL has an amino acid sequence encoded by a nucleic acid sequence within the nucleic acid sequence of the insert of the plasmid deposited with the ATCC having ATCC accession number PTA-127519.

[0215] The present invention also encompasses fusion proteins comprising one or more components of the antibodies disclosed herein. In some embodiments, fusion proteins can be made that include all or a portion of the anti-PD1 antibody of the present invention linked to another polypeptide. In another embodiment, only the variable domain of the anti-PD1 antibody is linked to the polypeptide. In another embodiment, the VH domain of the anti-PD1 antibody is linked to a first polypeptide, while the VL domain of the anti-PD1 antibody is linked to a second polypeptide that associates with the first polypeptide in such a way that the VH and VL domains can interact with each other to form an antigen-binding site. In another embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the polypeptide of interest. Further, fusion antibodies can be made in which two (or more) single-chain antibodies are linked to each other. This is useful when it is desired to create a bivalent or multivalent antibody on a single polypeptide chain, or when it is desired to create a bispecific antibody.

[0216] IL-12 variant fusion protein In some embodiments, provided herein are IL-12 variants linked to another protein, which are referred to herein as "IL-12 variant fusion proteins".

[0217] In some embodiments, the IL-12 variant can be linked to various types of proteins, such as an antibody, another cytokine, an enzyme, or an antibody Fc domain.

[0218] The IL-12 variants provided herein can be linked to another protein in any suitable manner. For example, the IL-12 variant can be covalently linked to another protein directly (e.g., such that the amino acids of IL-12 are directly covalently linked to the amino acids of another protein), or via a polypeptide linker. Further, the IL-12 can be linked to another protein in any orientation. For example, it can be linked to the N or C terminus of another protein; similarly, either the N or C terminus of an IL-12 subunit can be linked to another protein.

[0219] When IL-12 is linked to another protein, one or both of the IL-12 subunits can be linked to the other protein. For example, when an IL-12 variant provided herein is linked to an antibody, in some embodiments, one subunit of IL-12 (e.g., p35) can be linked to the first chain of the antibody, and the other subunit of IL-12 (e.g., p40) can be linked to the second chain of the antibody. In other embodiments, only one of the IL-12 subunits (p35 or p40) is linked to the chain of the antibody, and the other unlinked IL-12 subunit associates in the complex by its interaction with the linked IL-12 subunit.

[0220] In some embodiments, when IL-12 is linked to another protein, the IL-12 can be prepared as a single polypeptide chain containing both the p35 and p40 subunits of IL-12. Incorporating the sequences of the p35 and p40 subunits into a single polypeptide that can assemble as an intact IL-12 molecule may be desirable to facilitate the expression of the recombinant IL-12 variants provided herein.

[0221] In some embodiments, the IL-12 variant is linked to an antibody. The linkage of the IL-12 variant to the antibody can be useful for one or more purposes, such as 1) targeting IL-12 to the location of the antibody target (e.g., when the antibody binds to a cell surface receptor, the linkage of IL-12 to that antibody can target IL-12 to cells having that cell surface receptor) and 2) conjugating IL-12 activity to the therapeutic effect of the antibody when the antibody has a therapeutic effect.

[0222] In some embodiments, provided herein are fusion proteins that link the IL-12 variants and anti-PD1 antibodies provided herein (referred to herein as "IL-12 variant / anti-PD1 fusion proteins", "IL-12 variant / anti-PD1 molecules", etc.). In some embodiments, the anti-PD1 antibody is the anti-PD1 antibody provided herein.

[0223] High expression of PD1 is mainly observed on CD8-positive and CD4-positive tumor-infiltrating lymphocytes (TILs) and is enriched in the tumor microenvironment (TME) compared to circulating T cell subsets. This anatomical localization and cellular expression profile suggest that targeting IL-12 activity to PD1-positive (PD1+) cells can reduce systemic activity while still allowing for potent anti-tumor immunity. Overall, directing IL-12 activity from the periphery to the tumor microenvironment will enhance the anti-tumor effect of IL-12 in both PD(L)1 antagonist-naïve tumors and PD(L)1 antagonist-resistant tumors while reducing systemic toxicity.

[0224] PD1-positive cells (e.g., CD8-positive T cells and CD4-positive T cells) often contain the IL-12 receptor. Thus, binding of the antibody portion of the IL-12 variant / anti-PD1 fusion protein to PD1 on immune cells containing the IL-12 receptor brings the IL-12 variant in the fusion protein close to the IL-12 receptor on PD1-positive cells. In other words, the IL-12 variant / anti-PD1 fusions provided herein can bind to both 1) PD1 and 2) the IL-12 receptor on the same cell (e.g., a T cell). This is also referred to as "cis-targeting" of IL-12 (i.e., targeting the IL-12 variant to the same cell that is bound by the antibody linked to the IL-12 variant). Cis-targeting of the IL-12 variant to PD1-positive cells has several potential benefits. First, considering the low affinity of the IL-12 variant for the IL-12 receptor, an IL-12 variant with reduced affinity for the IL-12 receptor (e.g., the IL-12 variants provided herein) has low activity against cells that are positive for the IL-12 receptor but negative for PD1. Cells that are positive for the IL-12 receptor but negative (or have low amounts) of PD1 are most commonly cells in the circulation / periphery (i.e., outside the tumor microenvironment). Thus, a fusion protein containing an IL-12 variant with reduced affinity for the IL-12 receptor and an anti-PD1 antibody will have minimal activity and associated potential toxicity against peripheral cells that do not express PD1. Second, considering the binding of the IL-12 variant / anti-PD1 fusion molecule to PD1, an IL-12 variant with reduced affinity for the IL-12 receptor (e.g., the IL-12 variants provided herein) can still have effective activity against cells that are positive for the IL-12 receptor and positive for PD1.In this context, the IL-12 variant and the IL-12 receptor still interact strongly enough to produce the downstream effects of IL-12 that bind to the IL-12 receptor (e.g., increasing the cytotoxicity of CD8 T cells, promoting CD4 Th1 cell differentiation, inhibiting the function of regulatory T cells (Tregs), and increasing the expression of additional cytokines and chemokines, resulting in various anti-tumor effects such as the recruitment of immune cells, inhibition of angiogenesis, and inhibition of tumor growth). Thus, an anti-PD1 antibody bound to PD1 efficiently maintains the IL-12 variant near the IL-12 receptor. Thus, linking an anti-PD1 antibody to an IL-12 variant with reduced affinity for the IL-12 receptor "rescues" the activity of that IL-12 variant in the sense that the IL-12 variant has low or no activity against the IL-12 receptor unless the IL-12 variant is maintained physically close to the IL-12 receptor by being linked to an anti-PD1 antibody that binds to the PD1 molecule on the cell surface near the IL-12 receptor on the same cell surface.

[0225] 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 the potency and efficacy of the fusion protein. In some embodiments, the IL-12 variant / anti-PD1 fusion proteins provided herein have one, two, or all three of the following characteristics: 1) preferentially deliver IL-12 receptor stimulation driven by PD1-mediated avidity to PD1-positive cells; 2) exhibit an improved therapeutic index compared to a full agonist IL-12 molecule (e.g., a wild-type IL-12-Fc fusion molecule), and 3) bind to an epitope on PD1 that allows for simultaneous binding of a PD1 antagonist (e.g., an antibody that blocks the PD1 and PDL1 interaction) to PD1 such that PD1 antagonist activity is maintained when the IL-12 variant / anti-PD1 fusion protein binds to PD1.

[0226] Exemplary IL-12 variants / anti-PD1 fusion proteins include those shown in the Examples and described in the claims and embodiments of this specification.

[0227] In one embodiment, an IL-12 variant / anti-PD1 fusion protein comprising the following features is provided herein. 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, at the C-terminus of the chain, a linker sequence that connects to the N-terminus of the IL-12 variant p35 amino acid sequence so as to form a single continuous polypeptide containing the following components (in order from N-terminus to C-terminus): anti-PD1 heavy chain-linker sequence-IL-12 variant p35 sequence. To facilitate the heterodimerization of the two heavy chains, there is a "knob" mutation in the Fc of one heavy chain and a "hole" mutation in the Fc of the other heavy chain. The p40 subunit of the IL-12 variant is connected to the p35 subunit via a disulfide bond.

[0228] In one embodiment, the IL-12 variant / anti-PD1 fusion protein provided herein is a fusion protein containing an IL-12 H10 mutant protein and an anti-PD1 antibody, TPP-77658. This fusion protein is also referred to herein as the "H10658 fusion." There are a total of five separate polypeptides in the H10658 fusion: 1) an antibody heavy chain (not linked to the IL-12 polypeptide); 2) an antibody heavy chain linked to the p35 of the H10 IL-12 mutant protein; 3) an antibody light chain (copy 1); 4) an antibody light chain (copy 2); and 5) the p40 of the IL-10 IL-12 mutant protein. Of the five separate polypeptides, there are four different polypeptide sequences (there are 2 copies of the antibody light chain in the fusion protein; both light chains have the same amino acid sequence). The amino acid sequences of the polypeptides in the H10658 fusion are shown in Table 5 below.

[0229] [Table 5]

[0230] The amino acid sequence of the H10658 fusion shown in Table 5 contains the following annotated features. In the heavy chain TPP-77658 sequence (SEQ ID NO: 5), there are effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined). In the heavy chain TPP-77658 (SEQ ID NO: 25) fused to p35 of the H10 sequence, there are effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), mutations that form the "knob" for the knob-in-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence [SGGGGSGGGGSGGGG (SEQ ID NO: 27)] that connects the heavy chain of H10 and p35. The C-terminal lysine of SEQ ID NO: 5 is optional.

[0231] In one embodiment, the IL-12 variant / anti-PD1 fusion protein provided herein is a fusion protein containing an IL-12 H10 mutant protein and an anti-PD1 antibody TPP-76868. This fusion protein is also referred to herein as the "H10868 fusion." There are a total of five separate polypeptides in the H10868 fusion: 1) the antibody heavy chain (not linked to the IL-12 polypeptide); 2) the antibody heavy chain linked to p35 of the H10 IL-12 mutant protein; 3) the antibody light chain (copy 1); 4) the antibody light chain (copy 2); 5) p40 of the IL-10 IL-12 mutant protein. Among the five separate polypeptides, there are four different polypeptide sequences (there are 2 copies of the antibody light chain in the fusion protein; both 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.

[0232]

Table 6

[0233] The amino acid sequence of the H10868 fusion shown in Table 6 contains the following annotated features. In the heavy chain TPP-76868 sequence (SEQ ID NO: 15), effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined) are present. In the heavy chain TPP-76868 (SEQ ID NO: 26) fused to p35 of the H10 sequence, effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), mutations that form the "knob" for the knob-in-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence [SGGGGSGGGGSGGGG (SEQ ID NO: 27)] that connects the heavy chain of H10 and p35 are present. The C-terminal lysine of SEQ ID NO: 15 is optional.

[0234] Biological Activity of IL-12 Variant / anti-PD1 Fusion Protein In addition to binding to the epitope on PD1, the IL-12 variant / anti-PD1 fusion protein of the present disclosure can mediate biological activity. That is, the present disclosure specifically binds to PD1 and has at least one of the following detectable activities: (i) specifically binds to human PD1; (ii) specifically binds to cynomolgus PD1; (iii) inhibits tumor growth; (iv) increases STAT4 phosphorylation; (v) increases interferon (IFN) gamma expression, and includes an isolated IL-12 variant / anti-PD1 fusion protein that mediates at least one detectable activity selected from the group consisting of.

[0235] While not bound by a particular theory, administration of the IL-12 variant / anti-PD1 fusion protein provided herein to a subject can efficiently 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 systemic toxicity derived from IL-12. Also, PD1-positive T cells in the TME are known to be potent mediators of anti-tumor activity. This can enhance the IL-12 anti-tumor effect in both PD(L)1-naïve (i.e., not previously treated with an agent that blocks the interaction between PD1 and PDL1) and PD(L)1-resistant (i.e., previously treated with an agent that blocks the interaction between PD1 and PDL1) tumors while reducing systemic immune system activation and potential toxicity derived from 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).

[0236] Similarly, in the non-tumor microenvironment (i.e., peripheral or normal tissue), there are low levels of PD1-positive cells, and thus, the IL-12 variant / anti-PD1 fusion protein provided herein results in minimal activity and toxicity due to attenuation of IL-12 activity in the IL-12 variant and reduced PD1 binding due to the low number of PD1-positive cells.

[0237] 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 that are resistant to treatment with one or both of the PD1 and PDL1 (collectively "PD(L)1") inhibitors].

[0238] Engagement of the IL-12 receptor by IL-12 results in the induction of STAT4 phosphorylation (pSTAT4), leading to upregulation of the transcription of type 1 helper T cell (「Th1」)-associated genes such as IFN gamma, thereby enhancing the functional capacity of T cells. Since phosphorylation of STAT4 is the major activation step in the dimerization of the IL-12 receptor induced by IL-12, pSTAT4 can serve as a receptor-proximal readout of IL-12 activity, and IFN gamma can serve as a downstream readout of IL-12 activity.

[0239] Polynucleotides encoding an IL-12 variant, an anti-PD1 antibody, a fusion protein, and methods of manufacture The present disclosure also provides polynucleotides encoding any of these molecules, including portions and modified versions of the IL-12 variants, anti-PD1 antibodies, or fusion proteins provided herein. Also included are methods of making any of the IL-12 variants, anti-PD1 antibodies, and fusion proteins provided herein. The polynucleotides can be made by procedures known in the art and the proteins can be expressed.

[0240] The anti-PD1 antibody of interest (monoclonal or polyclonal) can be sequenced and then the polynucleotide sequence can be cloned into a vector for expression or propagation. Production of recombinant monoclonal antibodies in cell culture can be carried out by cloning antibody genes from B cells by means known in the art. See, for example, Tiller et al., 2008, J. Immunol. Methods, 329, 112; U.S. Patent No. 7,314,622.

[0241] In some embodiments, provided herein are polynucleotides or multiple polynucleotides comprising an array or multiple arrays encoding one or both of the p35 and p40 subunits of the IL-12 variants provided herein. In some embodiments, provided herein are polynucleotides or multiple polynucleotides comprising an array or multiple arrays encoding any one or more of the polypeptides of the IL-12 variant / anti-PD1 fusion proteins provided herein. In some embodiments, provided herein are polynucleotides or multiple polynucleotides comprising an array or multiple arrays encoding one or both of the heavy or light chain variable regions of the anti-PD1 antibodies provided herein. Polynucleotides encoding the IL-12 variant, antibody, or fusion protein of interest can be maintained in a vector within a host cell, and then the host cell can be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0242] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the p35 subunit of the IL-12 H10 variant, which encodes the amino acid sequence of SEQ ID NO: 3. In some embodiments, a polynucleotide comprising the 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.

[0243] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the p40 subunit of the IL-12 H10 variant, which 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.

[0244] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the VH of the anti-PD1 mAb TPP-77658, which 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.

[0245] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the VL of the anti-PD1 mAb TPP-77658, which encodes the amino acid sequence of SEQ ID NO: 8. In some embodiments, a 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.

[0246] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain of the H10658 fusion, which encodes the amino acid sequence of SEQ ID NO: 5. In some embodiments, a 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.

[0247] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain-H10 mutant protein p35 fusion of the H10658 fusion, which encodes the amino acid sequence of SEQ ID NO: 25. In some embodiments, a 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.

[0248] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 light chain of the H10658 fusion, which encodes the amino acid sequence of SEQ ID NO: 6. In some embodiments, a 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.

[0249] 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, which 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.

[0250] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the p35 subunit of the IL-12 H10 variant, which comprises the nucleotide sequence of SEQ ID NO: 44.

[0251] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the p40 subunit of the IL-12 H10 variant, which comprises the nucleotide sequence of SEQ ID NO: 45.

[0252] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the VH of the anti-PD1 mAb TPP-77658, which comprises the nucleotide sequence of SEQ ID NO: 46.

[0253] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the VL of the anti-PD1 mAb TPP-77658, which comprises the nucleotide sequence of SEQ ID NO: 47.

[0254] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain of the H10658 fusion, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 48.

[0255] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 heavy chain-H10 mutant protein p35 fusion of the H10658 fusion, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 49.

[0256] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the TPP-77658 light chain of the H10658 fusion, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 50.

[0257] In some embodiments, the present disclosure provides a polynucleotide encoding the amino acid sequence of the H10 mutant protein p40 fusion of the H10658 fusion, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 45.

[0258] [Table 7-1]

[0259] [Table 7-2]

[0260] In some embodiments, the disclosure provides a polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any IL-12 variant provided herein. In some embodiments, the disclosure provides a polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding variant H10 of IL-12, wherein 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 disclosure provides a polynucleotide or 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.

[0261] In some embodiments, the present disclosure provides a polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any anti-PD1 antibody provided herein. In some embodiments, the present disclosure provides a polynucleotide or 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 a polynucleotide or 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 a polynucleotide or 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 a polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6 and a 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 optional. In some embodiments, the present disclosure provides a polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16 and a 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 optional.In some embodiments, the present disclosure provides a polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding an isolated antibody that binds to PD1 and comprises a light chain comprising the amino acid sequence of SEQ ID NO: 43 and a heavy chain comprising 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 optional.

[0262] In some embodiments, the present disclosure provides a polynucleotide or a plurality of polynucleotides comprising one or more nucleotide sequences encoding any of the IL-12 variants / anti-PD1 fusion proteins provided herein. In some embodiments, the present disclosure provides an IL-12 variant / anti-PD1 fusion protein comprising 1) a heavy chain antibody, 2) a heavy chain antibody linked to the p35 subunit of the H10 IL-12 mutant protein, 3) a light chain antibody, and 4) the p40 subunit of the H10 IL-12 mutant protein, wherein the heavy chain antibody comprises the amino acid sequence of SEQ ID NO: 5, the heavy chain antibody linked to the p35 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 25, the light chain antibody comprises the amino acid sequence of SEQ ID NO: 6, and the p40 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 4, and provides a polynucleotide or a plurality of polynucleotides comprising one or more nucleotide sequences encoding the IL-12 variant / anti-PD1 fusion protein. In some embodiments, the present disclosure provides an IL-12 variant / anti-PD1 fusion protein comprising 1) a heavy chain antibody, 2) a heavy chain antibody linked to the p35 subunit of the H10 IL-12 mutant protein, 3) a light chain antibody, and 4) the p40 subunit of the H10 IL-12 mutant protein, wherein the heavy chain antibody comprises the amino acid sequence of SEQ ID NO: 15, the heavy chain antibody linked to the p35 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 26, the light chain antibody comprises the amino acid sequence of SEQ ID NO: 16, and the p40 subunit of the H10 IL-12 mutant protein comprises the amino acid sequence of SEQ ID NO: 4, and provides a polynucleotide or a plurality of polynucleotides comprising one or more nucleotide sequences encoding the IL-12 variant / anti-PD1 fusion protein.

[0263] In some embodiments, provided herein are polynucleotides comprising the nucleic acid sequence of the insert of a plasmid deposited with the ATCC, accession number PTA-127517, which encodes the TPP-77658 heavy chain of the H10658 fusion. In some embodiments, provided herein are polynucleotides comprising the nucleic acid sequence of the insert of a plasmid deposited with the ATCC, accession number PTA-127518, which encodes the TPP-77658 heavy chain-H10 mutant protein p35 fusion polypeptide of the H10658 fusion. In some embodiments, provided herein are polynucleotides comprising the nucleic acid sequence of the insert of a plasmid deposited with the ATCC, accession number PTA-127519, which encodes the TPP-77658 light chain of the H10658 fusion. In some embodiments, provided herein are polynucleotides comprising the nucleic acid sequence of the insert of a plasmid deposited with the ATCC, accession number PTA-127520, which encodes the H10 mutant protein p40 subunit of the H10658 fusion.

[0264] Further provided herein are polypeptides comprising the amino acid sequence encoded by the DNA insert of a plasmid deposited with the ATCC, accession number PTA-127517, which encodes the TPP-77658 heavy chain of the H10658 fusion. Also provided herein are polypeptides comprising the amino acid sequence encoded by the DNA insert of a plasmid deposited with the ATCC, accession number PTA-127518, which encodes the TPP-77658 heavy chain-H10 mutant protein p35 fusion of the H10658 fusion. Also provided herein are polypeptides comprising the amino acid sequence encoded by the DNA insert of a plasmid deposited with the ATCC, accession number PTA-127519, which encodes the TPP-77658 light chain of the H10658 fusion. Also provided herein are polypeptides comprising the amino acid sequence encoded by the DNA insert of a plasmid deposited with the ATCC, accession number PTA-127520, which encodes the H10 mutant protein p40 subunit of the H10658 fusion.

[0265] In some embodiments, an anti-PD1 antibody is provided herein that comprises a VH encoded by a portion of the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127517 and a VL encoded by a portion of the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127519. In some embodiments, an anti-PD1 antibody is provided herein that comprises a heavy chain encoded by the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127517 and a light chain encoded by the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127519. In some embodiments, an anti-PD1 antibody is provided herein that comprises a heavy chain encoded by the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127518 and a light chain encoded by the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127519. In some embodiments, an anti-IL-12 variant is provided herein that comprises a p35 subunit encoded by a portion of the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127518 and a p40 subunit encoded by the DNA insert of a plasmid deposited with the ATCC and having accession number PTA-127520.

[0266] One of ordinary skill in the art will understand that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides carry minimal homology to the nucleotide sequences provided herein. Nevertheless, polynucleotides that vary due to differences in codon usage frequency are specifically contemplated by the present invention. Further, alleles of the genes containing the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that have been altered as a result of one or more mutations in the nucleotides, such as deletions, additions, or substitutions. The resulting mRNA and proteins may or may not have an altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, or database sequence comparison.

[0267] In one embodiment, the VH and VL domains, or full-length HC or LC, are encoded by separate polynucleotides. Alternatively, both VH and VL, or HC and LC, are encoded by a single polynucleotide chain.

[0268] Polynucleotides that are complementary to any such sequences are also encompassed by the present disclosure. The polynucleotide may be single-stranded (coding or antisense), double-stranded, DNA (genomic, cDNA, or synthetic), or an RNA molecule. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one fashion, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may or may not be present within the polynucleotides of the present disclosure, and the polynucleotides may or may not be linked to other molecules or support materials.

[0269] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of ordinary skill in the art can use the sequences provided herein and commercially available DNA synthesizers to produce the desired DNA sequence.

[0270] To prepare polynucleotides using recombinant methods, as further discussed herein, a polynucleotide containing 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. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-conjugation, or electroporation. Once introduced, the exogenous polynucleotide can be maintained intracellularly as a non-integrating vector (such as a plasmid) or integrated into the host cell genome.

[0271] A suitable cloning vector can be constructed by standard techniques or selected from a number of cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended to be used, but useful cloning vectors will generally have one or more characteristics such as i) the ability to self-replicate, ii) a single target for a particular restriction endonuclease, or iii) the ability to carry a gene for a marker that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacteriophages such as pUC18, pUC19, Bluescript (e.g., pBS SK+), and its derivatives, 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.

[0272] An expression vector is further provided. An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the invention. By expression vector is meant that it must be replicable in the host cell, either as an episome or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenovirus, adeno-associated virus, retrovirus, cosmids, and the expression vectors disclosed in PCT Publication No. WO87 / 04462. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements such as a ribosome binding site, a translation initiation site, and a stop codon are also usually required.

[0273] Vectors containing the polynucleotide of interest can be introduced into host cells by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (e.g., if the vector is an infectious agent such as vaccinia virus). The choice of introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0274] The present invention also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating a gene encoding an antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtilis), and yeast (such as S. cerevisae, S. pombe, or K. lactis).

[0275] Furthermore, any number of commercially and non-commercially available cell lines that express a polypeptide or protein can be used in accordance with the present invention. One of ordinary skill in the art will understand that different cell lines may have different nutritional requirements or may require different culture conditions for optimal growth and polypeptide or protein expression, and can modify the conditions as needed.

[0276] Pharmaceutical Compositions In another embodiment, the present invention includes a pharmaceutical composition.

[0277] "Pharmaceutical composition" refers to a mixture of an IL-12 variant, anti-PD1 antibody, or fusion protein of the present invention and one or more excipients.

[0278] The pharmaceutical composition of the present invention may be in various forms. These include, for example, liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, and solid dosage forms such as lyophilized powders. The form depends on the intended mode of administration and therapeutic application.

[0279] Other excipients and modes of administration known in the pharmaceutical industry can also be used. The pharmaceutical composition of the present invention can be prepared by any of the 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 are described in standard textbooks. Pharmaceutical formulations of drugs are described, for example, in Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al. (eds.), Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al. (eds.), Handbook of Pharmaceutical Excipients (3rd Edition), American Pharmaceutical Association, Washington, 1999.

[0280] Excipients that are acceptable are non-toxic to the recipient at the dosages and concentrations employed, and include buffering agents such as phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0281] Therapeutic, diagnostic, and other methods The IL-12 variants, anti-PD1 antibodies, and IL-12 variant / anti-PD1 fusion proteins of the invention are useful in a variety of applications including, but not limited to, as agents for therapeutic treatment methods and diagnostic methods.

[0282] In some embodiments, the IL-12 variants and IL-12 variant / anti-PD1 fusion proteins provided herein can be used to treat a subject for any condition in which an increase in IL-12 activity is beneficial. The IL-12 variants and IL-12 variant / anti-PD1 fusion proteins provided herein are particularly useful for situations where it is desirable to provide IL-12 activity to a subject in a tightly controlled or limited manner.

[0283] In one aspect, the present invention provides a method for treating cancer. In some embodiments, a method of treating cancer in a subject comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising any one of the IL-12 variants, antibodies, or fusion proteins described herein. In some embodiments, provided is a method of treating cancer in a subject, the method 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.

[0284] In some embodiments, provided herein is a method of inhibiting the growth of PD1 / PD-L1 treatment-resistant cancer cells in a subject, the method 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.

[0285] In some embodiments, provided herein is a method of promoting the infiltration of CD8 positive (+) T cells in the tumor microenvironment of a subject, the method 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.

[0286] In some embodiments, provided herein is a method of promoting STAT4 phosphorylation in a subject in need thereof, the method comprising administering to the 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.

[0287] In some embodiments, provided herein is a method of promoting the production of interferon gamma (IFNg) in a subject in need thereof, the method comprising administering to the 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.

[0288] In another aspect, the present invention further provides an IL-12 variant, an IL-12 variant / anti-PD1 fusion protein or a related pharmaceutical composition described herein for use in the described method of treating cancer. The present invention also provides the use of an IL-12 variant or an IL-12 variant / anti-PD1 fusion protein described herein in the manufacture of a medicament for treating cancer.

[0289] In some embodiments, cancers that can be treated with the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein include, for example, solid tumors or liquid tumors. In some embodiments, solid tumors for treatment with the 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, cancers that can be treated include bladder cancer, breast cancer, clear cell renal carcinoma, head and neck squamous cell carcinoma (HNSCC) [squamous cell carcinoma of the head and neck (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 cancer, 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 lymphoblastic leukemia, colorectal cancer (CRC), glioblastoma, uterine cancer, cervical cancer, penile cancer, gastric cancer (GC), and one or more of non-melanoma skin cancers. In some embodiments, cancers that can be treated with the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein include, for example, NSCLC previously treated with platinum-based treatment 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.

[0290] Administration and Dosage Typically, the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein of the present invention is administered in an amount effective to treat the conditions described herein. The molecules of the present invention can be administered as the molecule itself or, alternatively, as a pharmaceutical composition containing the molecule.

[0291] The molecules of the present invention are administered in an effective dosage for the intended treatment by any suitable route, in a form of a pharmaceutical composition adapted to such route.

[0292] In some embodiments, the antibody can be administered parenterally, for example, directly into the bloodstream, into muscle, or into an organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intracardiac, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques. In some embodiments, the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein provided herein is administered subcutaneously (SC). In some embodiments, the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein provided herein is administered intravenously (IV).

[0293] The dosage regimen for the antibodies of the present invention or compositions containing said antibodies is based on various 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 particular antibody being used. Thus, the dosage regimen may vary widely. In one embodiment, the total daily dosage of the antibody of the present invention for the treatment of the indicated conditions discussed herein is typically about 0.01 to about 100 mg / kg (i.e., the number of mg of the antibody of the present invention per kg of body weight). In another embodiment, the total daily dosage 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.

[0294] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein are 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).

[0295] 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.

[0296] 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.

[0297] The toxicity and efficacy of the prophylactic and / or therapeutic protocols of the present invention can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine LD 50 (the dose lethal to 50% of the population) and ED 50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 . Preferred prophylactic and / or therapeutic agents exhibit a high therapeutic index.

[0298] Co-administration 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 of the uses, methods or compositions defined herein, wherein the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein of the present invention is used in combination with one or more other therapeutic agents considered herein.

[0299] Administration of two or more "combined" agents means that all agents are administered in a time close enough to effect treatment of the subject. The two or more agents can be administered simultaneously or sequentially. Further, co - administration can be effected by mixing the agents prior to administration or by administering the agents at the same time but in different dosage forms at the same or different administration sites.

[0300] In some embodiments, the IL - 12 variants, anti - PD1 antibodies, or IL - 12 variant / 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 anti - cancer agent. These include, but are not limited to, vaccines, CAR - T cell - based therapies, radiation therapy, cytokine therapy, CD3 bispecific antibodies, inhibitors of other immunosuppressive pathways, inhibitors of angiogenesis, T - cell activators, inhibitors of metabolic pathways, mTOR inhibitors, inhibitors of the adenosine pathway, tyrosine kinase inhibitors such as, but not limited to, Inlyta, ALK inhibitors and sunitinib, BRAF inhibitors, epigenetic modifiers, IDO1 inhibitors, JAK inhibitors, STAT inhibitors, cyclin - dependent kinase inhibitors, biologic agents (including, but not limited to, antibodies against 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), and the like, including, but not limited to, administration of biologic agents and / or chemotherapeutic agents.

[0301] Examples of biologic agents include therapeutic antibodies, immunomodulators, and therapeutic immune cells.

[0302] Therapeutic antibodies may have specificity for a variety of different antigens. For example, the therapeutic antibody can be directed against a tumor-associated antigen such that binding of the antibody to the antigen promotes the death of cells expressing the antigen. In other examples, the therapeutic antibody can be directed against an antigen on an immune cell (e.g., PD1) such that binding of the antibody prevents downregulation of the activity of cells expressing the antigen (thereby promoting the activity of cells expressing the antigen). In another example, the therapeutic activity can be directed against an antigen such that binding of the antibody to the antigen stimulates the target molecule containing the antigen (i.e., the antibody is an agonist antibody) to promote a desired activity. In some situations, a therapeutic antibody may function by multiple different mechanisms (e.g., it can do both i) promote the death of cells expressing the antigen and ii) prevent the antigen from causing downregulation of the activity of immune cells that contact cells expressing the antigen).

[0303] Therapeutic antibodies can be directed against, for example, the antigens listed below. For some antigens, exemplary antibodies directed against the antigen are also included below (in square brackets / parentheses after the antigen). The following antigens may be referred to herein as "target antigens" and the like. Target antigens for the therapeutic antibodies herein include, for example, 4-1BB (e.g., utomilumab); 5T4; A33; alpha-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); carbonic anhydrase 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, obinutuzumab, ofatumumab, rituximab, ublituximab); CD22 (inotuzumab ozogamicin, moxetumomab pasudotox); CD25; CD28; CD30 (e.g., brentuximab vedotin); CD33 (e.g., gemtuzumab 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., lobaplizumab 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, semaprimab, genolimzumab (CBT-501), MEDI0680, nivolumab, pembrolizumab, RN888 (see WO2016 / 092419), sintilimab, spartalizumab, STI-A1110, tislelizumab, TSR-042]; PD-L1 (e.g., atezolizumab, durvalumab, BMS-936559 (MDX-1105), or LY3300054); PDGFRA (e.g., orlatumumab); plasma cell antigen; polySA; PSCA; PSMA; PTK7 [e.g., PD-06647020 (see US9409995)]; Ror1; SAS; SCRx6; SLAMF7 (e.g., elotuzumab); SHH; SIRPa (e.g., ED9, Effi-DEM); STEAP; TGF-beta; TIGIT; TIM-3; TMPRSS3; TNF-alpha precursor; TROP-2 (e.g., sacituzumab govitecan); TSPAN8; VEGF (e.g., bevacizumab, brolucizumab); VEGFR1 (e.g., ranibizumab); VEGFR2 (e.g., ramucirumab, ranibizumab); Wue-1 etc.

[0304] 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 format. For example, the therapeutic antibodies may have any format described elsewhere herein. In some embodiments, the therapeutic antibody may be a naked antibody. In some embodiments, the therapeutic antibody may be conjugated to a drug or other agent (also known as an “antibody-drug conjugate” (ADC)). In some embodiments, a therapeutic antibody against a particular antigen can be incorporated into a multispecific antibody (e.g., a bispecific antibody).

[0305] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein can be administered in combination with pattern recognition receptor (PRR) agonists, immunostimulatory cytokines, and cancer vaccines. There are multiple classes of PRR molecules, such as toll-like receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and stimulator of interferon genes (STING) proteins. Other PRRs include, for example, DNA-dependent activator of IFN regulatory factors (DAI) and Absent in Melanoma 2 (AIM2). In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein can be administered in combination with a TLR agonist (e.g., a TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist).

[0306] Examples of immunostimulatory cytokines useful in the methods, agents, and uses of the invention include GM-CSF, G-CSF, IFN-alpha, IFN-gamma; IL-2 (e.g., denileukin diftitox), IL-6, IL-7, IL-11, IL-15, IL-18, IL-21, and TNF-alpha.

[0307] Examples of cancer vaccines useful in the treatment methods, agents, and uses of the present invention include, for example, Sipuleucel-T and talimogene laherparepvec (T-VEC).

[0308] Examples of immunotherapy useful in the treatment methods, agents, and uses of the present invention include, for example, tumor-infiltrating lymphocytes (TIL) and chimeric antigen receptor T cells (CAR-T cells).

[0309] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially, bullatacin and bullatacinone); camptothecin (including synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobemycin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin gamma 1I and calicheamicin phi I1, see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183~186 (1994); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related pigment protein engyin antibiotics chromophore), actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophyllin, chromomycin, 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, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin, etc.; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU), etc.; folic acid analogs, for example, denopterin, methotrexate, pteropterin, trimethoprim, etc.; purine analogs, for example, fludarabine, 6-mercaptopurine, thiampurine, thioguanine, etc.; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, etc.; androgens, for example, calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenal agents, for example, aminoglutethimide, mitotane, trilostane, etc.; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; dephosphamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, for example, maytansine and ansamitocin, etc.; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;Roxanthrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; lysoxine; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2’,2”-trichloro-triethylamine; trichothecene (especially, T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitocin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid, for example, paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, for example, cisplatin and carboplatin, etc.; 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 above. Also, antihormonal agents that act to regulate or inhibit the hormonal action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERM) including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that act to regulate estrogen production in the adrenal gland by inhibiting aromatase enzyme, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and antiandrogens, such as, for example, flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin, etc.; KRAS inhibitor; MCT4 inhibitor; MAT2a inhibitor; tyrosine kinase inhibitors, such as, for example, sunitinib, axitinib, etc.; alk / c-Met / ROS inhibitors, such as, for example, crizotinib, lorlatinib, etc.;mTOR inhibitors, such as temsirolimus, gedatolisib, etc.; src / abl inhibitors, such as bosutinib; cyclin-dependent kinase (CDK) inhibitors, such as palbociclib, PF-06873600, etc.; erb inhibitors, such as dacomitinib, etc.; PARP inhibitors, such as talazoparib, etc.; SMO inhibitors, such as glasdegib, PF-5274857, etc.; EGFR T790M inhibitors, such as PF-06747775, etc.; EZH2 inhibitors, such as PF-06821497, etc.; PRMT5 inhibitors, such as PF-06939999, etc.; TGFRβr1 inhibitors, such as PF-06952229, etc.; and pharmaceutically acceptable salts, acids or derivatives of any of the above are also included. In certain embodiments, such additional therapeutic agents are bevacizumab, cetuximab, sirolimus, panitumumab, 5-fluorouracil (5-FU), capecitabine, tivozanib, irinotecan, oxaliplatin, cisplatin, trifluridine, tipiracil, leucovorin, gemcitabine, regorafenib or erlotinib hydrochloride.;

[0310] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein are administered in combination with a PD1 or PDL1 inhibitor. PD1 and PDL1 inhibitors are collectively referred to herein as "PD(L)1" inhibitors. In some embodiments, the PD(L)1 inhibitor is sasanlimab.

[0311] In some embodiments, the PD(L)1 inhibitor is an anti-PD1 or anti-PD-L1 antibody. These bind to PD1 or PDL1 and block the interaction between PD1 and PDL1. Examples of PD(L)1 inhibitors useful in the methods, agents and uses of the present invention include, for example, sasanalimab (also known as RN888, anti-PD1 IgG4 monoclonal antibody), pembrolizumab (also known as MK-3475, anti-PD1 IgG4 monoclonal antibody), nivolumab (also known as BMS-936558 or MDX1106, anti-PD1 IgG4 monoclonal antibody), semiprimab (also known as REGN-2810, anti-PD1 antibody), atezolizumab (also known as MPDL3280A, IgG1 engineered anti-PDL1 antibody), BMS-936559 (fully human anti-PDL1 IgG4 monoclonal antibody), MEDI4736 (also known as durvalumab, engineered IgG1 kappa anti-PDL1 monoclonal antibody having three mutations in the Fc domain to remove antibody-dependent cell-mediated cytotoxic activity). Further exemplary PD(L)1 inhibitors useful in the methods, agents and uses of the present invention include SHR1210 (anti-PD1 antibody), KN035 (anti-PDL1 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) described in Yang et al., Med.Res.Rev. (2019), 39, 265-301.

[0312] In some settings, it may be advantageous to combine the IL-12 variant / anti-PD1 fusion protein provided herein with an anti-PD1 antibody that binds to an epitope on PD1 that is different from the anti-PD1 antibody of the IL-12 variant / anti-PD1 fusion protein. For example, some of the anti-PD1 antibodies provided herein bind to epitopes on PD1 such that the antibody does not block the interaction of PD1 with PDL1. In contrast, many or all of the anti-PD1 antibodies previously approved for therapeutic use bind to epitopes on PD1 such that the antibody inhibits the interaction of PD1 with PDL1.

[0313] Anti-PD1 antibodies that do not block the interaction of PD1 with PDL1 are useful for targeting the IL-12 variant / anti-PD1 fusion protein to PD1-expressing cells, such as T cells in the tumor microenvironment.

[0314] Thus, in some embodiments, provided herein are combination therapies 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 of PD1 with PDL1; and 2) a PD(L)1 inhibitor that blocks the interaction of PD1 with PDL1. In some embodiments, the PD(L)1 inhibitor is an anti-PD1 antibody that inhibits the interaction of PD1 with PDL1. In some embodiments, the PD(L)1 inhibitor is an anti-PDL1 antibody that inhibits the interaction of PD1 with PDL1.

[0315] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein are administered in combination with a VEGF or vascular endothelial growth factor receptor (VEGFR) inhibitor. VEGF and VEGFR inhibitors are collectively referred to herein as "VEGF(R)" inhibitors. VEGF(R) inhibitors include agents that bind to any VEGF subtype (e.g., VEGF-A, VEGF-C, and VEGF-D) as well as VEGFR subtypes (e.g., VEGFR1, VEGFR2, and VEGFR3). In some embodiments, the VEGF(R) inhibitor is axitinib or bevacizumab.

[0316] In some embodiments, the VEGF(R) inhibitor is an anti-VEGF or anti-VEGFR antibody. These bind to VEGF or VEGFR, block the interaction between VEGF and VEGFR, and / or inhibit the activity of VEGFR. Examples of anti-VEGF(R) antibodies include, for example, bevacizumab, ramucirumab, and ranibizumab. In some embodiments, the VEGF(R) inhibitor is a small molecule agent that binds to VEGF or VEGFR and inhibits the activity of VEGFR. Examples of small molecule VEGF(R) inhibitors include, for example, apatinib, axitinib, cabozantinib, lapatinib, lenvatinib, nintedanib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, and vandetanib.

[0317] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins provided herein may be co-administered with other drug treatments at intervals ranging from a few minutes to a few weeks, or administered sequentially before or after such other drug treatments. In embodiments where other drugs and / or proteins or polynucleotides are administered separately, generally, no significant period of time elapses between each delivery, such that the drugs and compositions of the present invention should still be able to exert an advantageously combined effect on the subject. In such cases, it is contemplated that both modalities can be administered within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other. In some situations, it may be desirable to greatly extend the period of administration, in which case, several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration.

[0318] In some embodiments, the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins are combined with a treatment regimen further comprising a traditional therapy selected from the group consisting of surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative care.

[0319] Kit Another aspect of the invention provides a kit comprising an IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein provided herein. The kit may further comprise one or more diagnostic or therapeutic agents in addition to the IL-12 variant, anti-PD1 antibody, or IL-12 variant / anti-PD1 fusion protein. The kit may also comprise instructions for use for use in a diagnostic or therapeutic method. In some embodiments, the kit comprises an antibody or a pharmaceutical composition thereof and a diagnostic agent. In other embodiments, the kit comprises 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).

[0320] In yet another embodiment, the invention includes a kit suitable for use in the execution of the treatment methods described herein. In one embodiment, the kit contains a first dosage form comprising an amount sufficient to carry out the method of the invention of one or more of the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins of the invention. In another embodiment, the kit includes an amount sufficient to carry out the method of the invention of one or more of the IL-12 variants, anti-PD1 antibodies, or IL-12 variant / anti-PD1 fusion proteins of the invention and a first container for at least a first dosage amount and a second container for a second dosage amount.

[0321] Biological Deposit Representative materials of the present invention were deposited on February 7, 2023, with 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 encoding the TPP-77658 heavy chain of the H10658 fusion. The vector "HCp35_H10658" with ATCC accession number PTA-127518 contains a DNA insert encoding the TPP-77658 heavy chain-H10 mutant protein p35 fusion of the H10658 fusion. The vector "LC_H10658" with ATCC accession number PTA-127519 contains a DNA insert encoding the TPP-77658 light chain of 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.

[0322]

Table 8

[0323] The deposit was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty). This ensures the maintenance of viable cultures of the deposited material for 30 years from the date of deposit. The deposited material is subject to the terms of the Budapest Treaty, as well as the contract between Pfizer, Inc. and the ATCC, and will be made available by the ATCC under the contract to guarantee the permanent and unrestricted availability of progeny cultures of the deposited material to the public, either upon the issuance of the relevant U.S. patent or, whichever is earlier, upon the publication of the U.S. or foreign patent application to the public, and to guarantee the availability of progeny to those determined by the Commissioner of the U.S. Patent and Trademark Office to have the right under 35 U.S.C. § 122 and the Commissioner's regulations thereunder (including 37 C.F.R. § 1.14, specifically referring to 886 OG 638).

[0324] The agent of this application agrees to immediately replace the deposited material with another identical one if notified that the cultures of the deposited material have died, been lost, or been destroyed when cultured under suitable conditions. The availability of the deposited material should not be construed as a license to practice the invention in violation of the rights granted under the patent laws of any government authority.

[0325] 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.

[0326] Summary of Sequences The sequences provided in this application are summarized in Table 9 below.

[0327]

Table 9-1

[0328]

Table 9-2

[0329]

Table 9-3

[0330]

Table 9-4

[0331]

Table 9-5

[0332]

Table 9-6

[0333]

Table 9-7

[0334] The above description and the following examples detail certain specific embodiments of the present disclosure and describe the best mode contemplated by the inventors. However, even if the above is presented in great detail in the text, it will be understood that the present disclosure can be implemented in many ways and that the present disclosure should be construed in accordance with the appended claims and any equivalents thereof.

[0335] The disclosed teachings have been described with reference to various uses, methods, kits, and compositions, but it will be understood that various changes and modifications can be made without departing from the teachings of this specification and the following claimed disclosure. The above 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 from the perspective of these exemplary embodiments, those skilled in the art will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

Example

[0336] To better understand the present invention, the following examples are described. These examples are merely illustrative and should not be construed as limiting the scope of the present invention in any way.

[0337] (Example 1) IL-12 Variant The purpose of this experiment was to generate a human IL-12 variant (also referred to as an IL-12 mutant protein or "mutant protein") having attenuated IL-12 activity compared to wild-type human IL-12.

[0338] A set of IL-12 variants was designed using mutations engineered in one or both of the p35 and p40 subunits of human IL-12. The IL-12 variants are described in Table 10. In Table 10, the positions of the mutations are numbered based on the amino acid sequences of the mature human p35 and p40 proteins (SEQ ID NO: 1 and SEQ ID NO: 2, respectively). As shown in Table 10, the mutations were at one or more positions of F39, I52, and Y167 in the p35 subunit, and at one or more positions of D93 and K85 in the p40 subunit. These amino acid positions are predicted to be located at the interface between IL12 and the IL12 receptor.

[0339]

Table 10

[0340] Fusion proteins were prepared in which different IL-12 mutant proteins shown in Table 10 were linked to an anti-human PD1 antibody as a platform. Subsequently, the activities of the different IL-12 mutant protein / anti-PD1 fusion proteins were evaluated.

[0341] First, the activity of the IL-12 mutant protein / 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 ) for the fusion protein is shown in the column of "EC50 hIL12R+ cells" in Table 10 together with this cell line (evaluating STAT4 phosphorylation). EC 50 is provided in micrograms per milliliter (μg / ml). A lower EC 50 value indicates higher activity than a higher EC 50 value.

[0342] Next, the activity of the IL-12 mutant protein / anti-PD1 fusion protein was evaluated using an IL-12 receptor-positive (IL12R+), human PD1-positive pSTAT4 reporter cell line. The half-maximal effective concentration (EC 50 ) for the fusion protein is shown in the column of "EC50 hIL12R+PD1+ cells" in Table 10 together with this cell line (evaluating STAT4 phosphorylation). This data provides information on the PD1-induced and rescued IL-12 mutant protein activity of each fusion protein (e.g., by comparing the activities of each fusion protein between the PD1-negative and PD1-positive cell lines).

[0343] As shown in Table 10, multiple IL-12 mutant proteins / anti-PD1 fusion proteins have low activity in hIL12R+ and PD1-negative cells, but higher activity in hIL12R+ and PD1-positive cells (e.g., mutant protein H10). Thus, these fusion proteins have targeted IL-12 activity against PD1-positive cells.

[0344] As further shown in Table 10, the mutant protein "H10" is indeterminate but has low activity (EC higher than 3000 μg / ml 50 ; the highest concentration tested) on PD1-negative cells, but higher activity (EC of 686 μg / ml 50 ) on PD1-positive cells.

[0345] 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 on PD1-negative cells. The lack of activity on PD1-negative cells potentially limits the number of cells that can be efficiently stimulated by the H10 IL-12 mutant protein and thus potentially reduces the toxicity associated with IL-12 activity (e.g., caused by overstimulation mediated by IL-12 of the immune response). Second, the H10 mutant protein linked to the anti-PD1 antibody has low activity on PD1-negative cells, but the H10 IL-12 mutant protein / anti-PD1 fusion protein still has detectable activity on PD1-positive cells. Thus, the H10 IL-12 mutant protein / anti-PD1 fusion protein is hypothesized to still be active in environments enriched for PD1-positive cells such as the tumor microenvironment (TME). Third, evaluation of the biophysical properties (e.g., stability and predicted immunogenicity) of the H10 mutant protein showed that the H10 mutant protein has more desirable molecular properties than other IL12 mutant proteins that also have activity biased towards PD1-positive cells compared to PD1-negative cells when linked to the anti-PD1 antibody.

[0346] The H10 IL12 mutant protein contains the mature p35 amino acid sequence of SEQ ID NO: 3 and the mature p40 amino acid sequence of 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 mutations D93L (underlined) and also a mutation in the heparin-binding site GGG (underlined). For the heparin-binding site mutation, the amino acid sequence KSKREKK (SEQ ID NO: 30) (amino acids 258 - 264 in SEQ ID NO: 4) in the wild-type IL-12p40 sequence is mutated and truncated downward to the sequence "GGG". Wild-type IL-12 is known to bind to heparin and heparan sulfate (Hasan M. et al., J Immunol. 1999 Jan 15;162(2):1064 - 70); the 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. The binding of IL-12 to heparin is hypothesized to enhance IL-12 function (e.g., by retaining IL-12 near the site of secretion, acting to maintain a high local cytokine concentration). Furthermore, the heparin-binding site in IL-12 is protease-sensitive; thus, removal of that site reduces the protease sensitivity of the H10 mutant protein.

[0347] H10 mutant protein p35: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDF A KTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 3)

[0348] H10 mutant protein p40: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWST L ILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQG GGG DRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 4)

[0349] (Example 2) Non-blocking anti-PD1 antibody Using a multi-step process, new non-blocking anti-human PD1 (hPD1) antibodies were generated and selected. The generated anti-hPD1 antibodies include clone GBT-PD1-0009, GBT-PD1-0013, and GBT-PD1-0017.

[0350] A non-blocking anti-hPD1 antibody is an antibody that does not bind to the same epitope on hPD1 that is bound by human PDL1 (hPDL1), the hPD1 ligand. A non-blocking anti-hPD1 antibody does not prevent the binding of hPDL1 to hPD1. Furthermore, a non-blocking antibody does not prevent the binding of many currently available therapeutic antagonist anti-hPD1 antibodies (e.g., pembrolizumab, cemiplimab, nivolumab, etc.), which potentially inhibit the binding of hPDL1 to hPD1, to PD1.

[0351] To evaluate whether various antibodies can bind to hPD1 simultaneously, a sandwich assay was performed in which two different antibodies were incubated with hPD1 simultaneously. Further, in some assays, the antibodies were incubated with a fusion molecule (hPDL1-Fc) containing hPDL1 covalently linked to the antibody Fc domain to evaluate whether each antibody could bind to hPD1 simultaneously with hPDL1.

[0352] Table 11 shows the results of the sandwich assay. In Table 11, the corresponding positions in the table are listed with "Y" if the paired antibodies listed in the corresponding columns and rows can bind to hPD1 simultaneously (i.e., if there is no competition between the antibodies for binding to hPD1), or "N" if there is competition between the antibodies for binding to hPD1. As an example, as shown in Table 11, when nivolumab, pembrolizumab, or hPDL1 is present at each of these positions in the table (indicated by "N"), the binding of semiprimab to hPD1 is inhibited. In contrast, in the presence of GBT-PD1-0009, GBT-PD1-0013, or GBT-PD1-0017, the binding of semiprimab to hPD1 is not inhibited.

[0353]

Table 11

[0354] For each of GBT-PD1-0009, GBT-PD1-0013, or GBT-PD1-0017, the binding of each antibody to PD1 is not inhibited by hPDL1, semiprimab, 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 at the epitope bound by semiprimab, nivolumab, or pembrolizumab.

[0355] To verify the non-blocking nature of GBT-PD1-0013, the X-ray crystal structure of GBT-PD1-0013 complexed with the extracellular domain (ECD) of human PD1 was elucidated. The crystal structure showed that GBT-PD1-0013 binds to human PD1 ECD in a 1:1 stoichiometry at a site different from that of the natural ligand PD-L1 when determined by aligning the relevant complex structures.

[0356] 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 readouts using the BW5147.3 (T lymphoblast) cell line overexpressing human PD1. To enable flow cytometry-based readouts, PD1(B) and GBT-PD1-0013 were conjugated to fluorescent dyes (AlexaFluor488 and AlexaFluor647, respectively). A fixed number of cells were incubated with i) individual antibodies 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 FIGS. 1A and 1B. In these figures, GBT-PD1-0013 is referred to as “PD1(NB)” and the blocking anti-PD1 antibody is referred to as “PD1(B)”. FIGS. 1A and 1B show that the binding of GBT-PD1-0013 to cells expressing PD1 was minimally affected by the presence of PD1(B) and vice versa. Specifically, FIG. 1A shows that the binding of GBT-PD1-0013 to cells expressing PD1 was minimally affected by the presence of PD1(B) (i.e., the % of cells expressing PD1 bound by GBT-PD1-0013 decreased only slightly when incubated with GBT-PD1-0013 and PD1(B) together with cells expressing PD1 compared to when incubated with GBT-PD1-0013 alone with cells expressing PD1). Similarly, FIG. 1B shows that the binding of PD1(B) to cells expressing PD1 was minimally affected by the presence of GBT-PD1-0013.

[0357] Clone GBT-PD1-0013 was modified to generate a series of related non-blocking anti-hPD1 antibodies that had various affinities for hPD1 and reduced predicted immunogenicity (based on fewer predicted T cell epitopes) compared to the parental GBT-PD1-0013 antibody. The binding characteristics of different clones to hPD1 are provided in Table 12.

[0358]

Table 12

[0359] (Example 3) IL12 mutant protein - non-blocking anti-PD1 antibody fusion protein This example describes the preparation and characterization of a fusion protein that combines an IL-12 H10 mutant protein (Example 1) with various non-blocking anti-PD1 antibodies (Example 2).

[0360] A plurality of non-blocking anti-PD1 antibodies 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-12 mutant protein H10 was connected to the C-terminus of one heavy chain of the antibody via a linker having the amino acid sequence: SGGGGSGGGGSGGGG (SEQ ID NO: 27) (the p40 subunit of the IL-12 H10 mutant protein associates with the antibody via its interaction with the p35 subunit). A general schematic of the fusion protein is shown in Figure 2. As shown in Figure 2, one of the heavy chains of the anti-human PD1 antibody is covalently linked to the p35 subunit of the IL-12 mutant protein via a linker; the p40 subunit of IL-12 associates with p35 via a disulfide bond between C74 of p35 and C177 of p40. Furthermore, to promote heterodimerization between 1) the heavy chain of the antibody covalently linked to p35 and 2) the heavy chain not linked to p35, each heavy chain has a mutation in the Fc region that forms a knob or hole structure. As depicted in Figure 2, the heavy chain of the antibody covalently linked to p35 has a mutation that forms a knob, and the heavy chain not linked to p35 has a mutation that forms a hole. Furthermore, both heavy chains have a mutation in the Fc domain that renders the Fc effector null. The antibody is of the IgG1 subclass.

[0361] The fusion protein was prepared using an inactivated Fc domain to remove potential Fc-mediated depletion and / or Fc receptor binding, and further to concentrate the binding and activity of IL-12 towards PD1-positive cells.

[0362] Overall, the anti-PD1 portion of the IL-12 H10 mutant protein / anti-PD1 fusion protein serves to "anchor" the fusion protein to cells that are PD1-positive and IL-12 receptor-positive; thus, the anti-PD1 portion of the fusion protein serves to concentrate IL12 activity towards cells that are double-positive for both PD1 and the IL-12 receptor.

[0363] The activities of these fusion proteins were evaluated using human primary cells derived from healthy peripheral blood mononuclear cell (PBMC) donors. After purified CD4 T cells were activated in vitro, they were stimulated with different IL-12 H10 mutant protein / anti-PD1 fusion proteins to evaluate the level of phosphorylation of STAT4, which is a readout for IL-12 activity (activated CD4 T cells have increased levels of the IL-12 receptor and PD1).

[0364] The half maximal effective concentration (EC 50 ) for the fusion protein is shown in Table 13. Each data column relates to data from an individual PBMC donor.

[0365]

Table 13

[0366] As shown in Table 13, the EC 50 of the H10 / anti-PD1 fusion molecule is lower than that of H10 / isotype IgG, indicating that the H10 / anti-PD1 fusion molecule has target activity against PD1-positive cells.

[0367] The amino acid sequence of the polypeptide of the H10 / TPP-76868 fusion protein is provided in Table 14 below. This fusion is also referred to herein as the "H10868" fusion.

[0368] [Table 14]

[0369] 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 the Fc: L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined). In the heavy chain TPP-76868 (SEQ ID NO: 26) fused to p35 of the H10 sequence, there are effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), mutations that form the "knob" for the knob-in-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence [SGGGGSGGGGSGGGG (SEQ ID NO: 27)] that connects the heavy chain of H10 and p35.

[0370] IL-12 H10 mutant protein / anti-PD1 TPP-77658 fusion protein The amino acid sequence of the polypeptide of the H10 / TPP-77658 fusion protein is provided in Table 15 below. This fusion is also referred to herein as the "H10658" fusion.

[0371] [Table 15]

[0372] The amino acid sequence of the H10658 fusion shown in Table 15 contains the following annotated features. In the heavy chain TPP-77658 sequence (SEQ ID NO: 5), effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), and mutations that form the "hole" for the knob-in-hole structure: S354C, T366S, L368A, and Y407V (EU numbering; underlined) are present. In the heavy chain TPP-77658 (SEQ ID NO: 25) fused to p35 of the H10 sequence, effector null mutations in the Fc: L234A, L235A, and G237A (EU numbering; underlined), mutations that form the "knob" for the knob-in-hole structure: Y349C and T366W (EU numbering; underlined), and a linker sequence [SGGGGSGGGGSGGGG (SEQ ID NO: 27)] that connects the heavy chain of H10 and p35 are present.

[0373] (Example 4) Mouse surrogate IL-12 variant / anti-PD1 fusion molecule This example describes the generation of a mouse surrogate IL-12 variant / anti-PD1 molecule and related experiments. The biology of IL-12 is mostly conserved in humans and mice. However, human IL-12 does not cross-react with the mouse IL-12 receptor and cannot be used in preclinical mouse models. Therefore, a mouse surrogate IL-12 variant was generated to further evaluate the activity of the IL-12 variant.

[0374] Two different murine surrogate IL-12 variants / anti-PD1 molecules were developed. Both the surrogate IL-12 variants / anti-PD1 molecules were selected as having a level of attenuation similar to that of the human H10 IL12 mutant protein and contain the same murine IL-12 mutant protein (described in Example 1). To facilitate production, the murine IL-12 mutant protein was prepared as a single polypeptide in which the IL-12 p40 and p35 subunits are covalently linked via a peptide linker (rather than expressing p40 and p35 as separate polypeptide chains). The sequence of the murine IL-12 mutant protein (containing the linked p40 and p35 subunits) is as follows: 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 beginning of the sequence), the p35 subunit is the C-terminal portion (i.e., the end of 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 portions of their respective polypeptides (i.e., do not contain a leader sequence). The mutant protein has the following mutations compared to wild-type mouse IL-12 (each mutation is underlined in the above sequence; amino acid numbers are based on the mature polypeptide sequence): p40: GGG (reduces heparin binding) [replaced mouse p40 residues RIQRKK (amino acid numbers 251-256) with the sequence GGG]; p35: K34E, H35F, and S37P (each mutation is for attenuation of IL-12 activity).

[0375] Two different murine surrogate IL-12 variant / anti-PD1 fusion proteins contained either the above-described murine IL-12 mutant protein and a PD1-blocking anti-mouse PD1 antibody, or a PD1-non-blocking anti-mouse PD1 antibody. Thus, the two murine surrogate IL-12 variant / anti-PD1 fusions were 1) a murine 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 murine 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"].

[0376] The activities of both the murine surrogate IL-12 mutant protein and the anti-PD1 fusion protein were compared in ex vivo CT26 tumor stimulation to evaluate whether there were differences in the activities of the molecules due to different anti-PD1 specificities (blocking vs. non-blocking). CT26 tumors (murine colon cancer) were harvested from mice implanted with CT26 cells. After processing the tumors to achieve a single cell suspension of tumor cells and other cells in the tumor microenvironment, they were stimulated with one of four different fusion proteins: 1) murine IL-12 wild type linked to the murine Fc domain (“mIL12 wt-Fc”); 2) murine IL-12 mutant protein linked to the murine Fc domain (“mIL12 mutant protein-Fc”); 3) mIL12 mutant protein-PD1(B); or 4) mIL12 mutant protein-PD1(NB). After 24 hours, the activities of the different fusion proteins were evaluated as measured by the amount of interferon gamma (IFNg) present in the supernatant after 24 hours.

[0377] The results are shown in Figure 3. In Figure 3, the data points are depicted as follows: 1) mIL12 wt-Fc: solid line, black squares; 2) mIL12 mutant protein-Fc: dashed line, star; 3) mIL12 mutant protein-PD1(B): solid line, triangles; or 4) mIL12 mutant protein-PD1(NB): solid line, white circles. As shown in Figure 3, the activities of mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB) were similar, suggesting that PD1 blockade is not required for IL12 mutant protein activity targeted by PD1. Figure 3 also shows that mIL12 wt-Fc has higher activity than mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB), which is consistent with the wild-type IL-12 activity of mIL12 wt-Fc. Figure 3 also shows that mIL12 mutant protein-Fc has lower activity than mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB), which is consistent with the lack of targeting of mIL12 mutant protein-Fc.

[0378] The activity of the human IL-12 variant / anti-PD1 fusion protein containing the H10 IL-12 mutant protein was compared with that of the murine surrogate IL-12 mutant protein / anti-PD1 fusion protein mIL12 mutant protein-PD1(B) in a matched assay set up 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 for this experiment contained the H10 IL-12 mutant protein (described in Example 1) linked to the non-blocking anti-hPD1 antibody TPP-68807 (described in Example 2; it is a derivative of GBT-PD1-0013 and is closely related to TPP-77658). This fusion protein was designated "hIL12 mutant protein-hPD1(NB)" in this experiment.

[0379] After activating CD4 T cells in vitro, they were stimulated with the following different fusion proteins. Human CD4 T cells were stimulated with 1) human IL-12 wild type conjugated to a human isotype (IgG1) control antibody (i.e., having no specificity for PD1) ("hIL12 wt-isotype"); 2) human H10 IL-12 mutant protein (described in Example 1) fused to a human isotype (IgG1) control antibody ("hIL12 mutant protein-isotype"); and 3) hIL12 mutant protein-hPD1 (NB). Mouse CD4 T cells were stimulated with 1) mouse IL-12 wild type fused to a human isotype (IgG1) control antibody (i.e., having no specificity for PD1) ("mIL12 wt-isotype"); 2) mouse IL-12 mutant protein corresponding to human H10 fused to a human isotype (IgG1) control antibody ("mIL12 mutant protein-isotype"); and 3) mIL12 mutant protein-PD1(B) (described above). For each assay, pSTAT4 was measured by flow cytometry 60 minutes after T cell stimulation. The assay included analysis of the estimated number of PD1 receptors per cell, and the pSTAT4 readout was limited to cells having a similar number of PD1 receptors per cell to enable more accurate comparison. The results are summarized in Table 16 below.

[0380]

Table 16

[0381] As shown in Table 16, both human and mouse IL12 mutant proteins have a similar level of attenuation of activity (a decrease to approximately 1 / 23,000) compared to their respective human or mouse wild-type IL12. Furthermore, the PD1-induced rescue of the activity of the IL12 mutant proteins (measured as the increase in activity of the IL12 mutant protein / anti-PD1 antibody fusion over the IL12 mutant protein / nonspecific isotype antibody fusion) is also similar for both human and mouse fusions (an increase of approximately 100-fold). Overall, this experiment shows that the human hIL12 mutant protein-hPD1(NB) fusion protein and the mouse surrogate mIL12 mutant protein-PD1(B) fusion protein are in exact agreement for both attenuation of IL12 and the rescued activity by the addition of the anti-PD1 antibody. Furthermore, considering the above experiment showing equivalent activity of the mIL12 mutant protein-PD1(B) and mIL12 mutant protein-PD1(NB) fusion proteins, it can be concluded that the hIL12 mutant protein-hPD1(NB) fusion protein has similar activity to both the mIL12 mutant protein-PD1(B) fusion protein and the mIL12 mutant protein-PD1(NB) fusion protein in their respective experimental systems.

[0382] (Example 5) In vivo efficacy test In two syngeneic mouse tumor models (MC38R and B16F10), efficacy tests were performed. MC38R is a mouse colon adenocarcinoma cell line. B16F10 is a mouse melanoma cell line. The molecules listed in Table 17 were used in these tests.

[0383]

Table 17

[0384] MC38R Experiment 1 Methods: 500,000 MC38R tumor cells were implanted subcutaneously into female C57BL / 6 mice. Ten days later, the mice were randomized into treatment groups with an average tumor volume of 44 - 92 mm 3 . The mice were treated with a single subcutaneous dose of 1) mIL12 mutant protein - PD1 (NB); 2) mIL12 mutant protein - isotype; 3) mouse isotype; or 4) mPD1 (B). On the day following randomization, mIL12 mutant protein - PD1 (NB) and mIL12 mutant protein - isotype were administered at 0.05 mg / kg, 0.17 mg / kg, or 0.5 mg / kg, and mouse isotype and mPD1 (B) were administered at 0.5 mg / kg. Measurements of tumor volume and body weight were taken twice weekly until the end of the study when the tumor volume of the first mouse reached 2000 m 3 . There were 10 mice per treatment group.

[0385] Results: In this experiment, single - dose subcutaneous administration of mIL12 mutant protein - PD1 (B) at 0.05 mg / kg, 0.17 mg / kg, or 0.5 mg / kg induced potent and dose - dependent tumor growth inhibition (TGI) (61%, 76%, and 92% respectively), whereas administration of mIL12 mutant protein - isotype at the same dose levels did not induce significant TGI (-25%, -13%, and 45% respectively). Furthermore, administration of the plain mPD1 (B) antibody (i.e., not fused to the IL12 mutant protein) also did not induce significant TGI (12%). The lack of TGI in the mPD1 (B) antibody treatment group was expected because it was administered at one - twentieth the amount and lower frequency (single - dose instead of 3 times every 3 days or 2 times weekly) of a therapeutically administered PD1 antagonist antibody. No significant body weight loss (BWL) was observed at any dose level tested for any of these molecules.

[0386] MC38R Experiment 2 Methods: 500,000 MC38R tumor cells were implanted subcutaneously into female C57BL / 6 mice. Ten days later, the mice were randomized into treatment groups with an average tumor volume of 31 - 131 mm 3The treatment groups were randomized based on the mean tumor volume. Mice were treated on the day after randomization with a single subcutaneous dose of 0.5 mg / kg of 1) mIL12 wt-isotype; 2) mIL12 wt-PD1(B); 3) mIL12 mutant protein-isotype; 4) mIL12 mutant protein-PD1(B); 5) mouse isotype or 6) mPD1(B). Tumor volume and body weight were measured twice weekly until the end of the study when the tumor volume of the first mouse reached 2000 m 3 There were 3 - 10 mice per treatment group. Measurements were taken twice weekly for tumor volume and body weight until the end of the study when the tumor volume of the first mouse reached 2000 m

[0387] Results: mIL12 mutant protein-PD1(B), mIL12 wt-isotype, and mIL12 wt-PD1(B) each induced potent and significant TGI when administered subcutaneously as a single dose of 0.5 mg / kg, compared to isotype-treated animals: 87% TGI for mIL12 mutant protein-PD1(B), 96% TGI for mIL12 wt-isotype, and 70% TGI for mIL12 wt-PD1(B). Treatment with mIL12 mutant protein-isotype did not induce significant TGI (14%). Mice treated with mIL12 mutant protein-PD1(B) did not show BWL, while mice treated with either mIL12 wt-isotype or mIL12 wt-PD1(B) showed significant BWL on day 6 post-administration, with mean BWL of 21% and 19% respectively.

[0388] These results indicate that mIL12 mutant protein-PD1(B) can induce potent TGI without BWL; this is in contrast to mIL12 wt-isotype and mIL12 wt-PD1(B), both of which induce potent TGI but also induce significant BWL.

[0389] MC38R Experiment 3 For this experiment, MC38R B2M KO cells were used. In these tumor cells, the B2M gene is deleted, and thus the tumor cells cannot load antigens onto major histocompatibility complex (MHC) I. As a result, the tumors become MHC I-low or negative and resistant to direct CD8 T cell killing. Therefore, these tumor cells are also resistant to PD(L)1 therapy.

[0390] Methods: 500,000 MC38R B2M KO tumor cells were implanted subcutaneously into female C57BL / 6 mice. Seven days later, the mice were randomized into treatment groups with an average tumor volume of 52 - 91 mm 3 Starting on the day of randomization (d0), the 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 three times every three days (Q3Dx3) at 10 mg / kg, mPD1 RMP1 - 14 was administered twice weekly (QWx2) at 10 mg / kg, and mouse isotype was administered QWx2 at 10 mg / kg. Tumor volume and body weight were measured twice weekly until the end of the study when the tumor volume of the first mouse reached 2000 m 3 . There were 10 mice per treatment group.

[0391] Results: The single dose of mIL12 mutant protein - PD1 (NB) induced a strong and significant TGI (75%) compared to mouse isotype. In contrast, mPD1 F2 and mPD1 RMP1 - 14 were unable to induce a significant TGI against these tumor cells (-23% and 5% respectively compared to mouse isotype). No BWL was observed in all groups.

[0392] These results indicate that mIL12 mutant - PD1 (NB) is effective in inducing TGI in a PD(L)1 - resistant tumor model.

[0393] B16F10 Experiment 1 Method: 500,000 B16F10 tumor cells were implanted subcutaneously into female C57BL / 6 mice. Ten days later, the mice were randomized into treatment groups with an average tumor volume of 52 - 111 mm 3 On the day of randomization (d0), the mice were treated with 1) mIL12 mutant protein - PD1 (B), 2) mIL12 mutant protein - PD1 (NB), or 3) a single subcutaneous dose of mouse isotype. mIL12 mutant protein - PD1 (B) and mIL12 mutant protein - PD1 (NB) were each administered subcutaneously once at 0.5 mg / kg and 1.5 mg / kg (different treatment groups), and mouse isotype was administered at 1.5 mg / kg.

[0394] Results: A single dose of mIL12 mutant protein - PD1 (B) induced potent, dose - dependent TGI (0.5 mg / kg: 67% TGI; 1.5 mg / kg: 87% TGI). Furthermore, a single dose of mIL12 mutant protein - PD1 (NB) also induced potent, dose - dependent TGI (0.5 mg / kg: 76% TGI; 1.5 mg / kg: 78% TGI). No BWL was observed in all groups.

[0395] These results indicate the efficacy of mIL12 mutant protein - PD1 (B) and mIL12 mutant protein - PD1 (NB) molecules in the induction of TGI in the B16F10 tumor model. Furthermore, these results indicate that the efficacy of mIL12 mutant protein - PD1 (B) and mIL12 mutant protein - PD1 (NB) molecules does not depend on the antagonism of the interaction between PD1 and PDL1.

Claims

**Claim 1** An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at position Y167 of SEQ ID NO: 1 (IL-12 p35 subunit) and an amino acid substitution at position D93 of SEQ ID NO: 2 (IL-12 p40 subunit). **Claim 2** The IL-12 variant according to claim 1, wherein the Y167 substitution is Y167A. **Claim 3** The IL-12 variant according to any one of claims 1 to 2, wherein the D93 substitution is D93L. **Claim 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. **Claim 5** The IL-12 variant according to any one of claims 1 to 4, wherein the p40 subunit further comprises one or more mutations that reduce the binding of IL-12 to heparin. **Claim 6** The IL-12 variant according to claim 5, wherein the mutations that reduce the binding of IL-12 to heparin include the substitutions K258G, S259G, and K260G of SEQ ID NO: 2, and the deletions of R261, E262, K263, and K264. **Claim 7** 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-12 p35 subunit) and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (variant IL-12 p40 subunit). **Claim 8** An isolated human interleukin 12 (IL-12) variant comprising an amino acid substitution at one or more positions selected from F39 of SEQ ID NO: 1 (IL-12 p35 subunit), I52 of SEQ ID NO: 1, Y167 of SEQ ID NO: 1, K85 of SEQ ID NO: 2 (IL-12 p40 subunit), and D93 of SEQ ID NO:

2. **Claim 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. **Claim 10** The IL-12 variant according to any one of claims 8 to 9, wherein the p40 subunit further comprises one or more mutations that reduce the binding of IL-12 to heparin. **Claim 11** An IL-12 variant according to any one of claims 1 to 10, having reduced activity as 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, 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.

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

8.

14. 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 optional.

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, 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.

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, 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.

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

18. An isolated fusion protein comprising the human interleukin 12 (IL-12) variant according to 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-12 p35 subunit) and an amino acid substitution at position D93 of SEQ ID NO: 2 (IL-12 p40 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), VH comprises the amino acid sequence of SEQ ID NO: 7, and 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, the fusion protein comprising the polypeptides of SEQ ID NO: 5, 25, 6, and 4.

23. An isolated fusion protein comprising a human interleukin 12 (IL-12) variant and an anti-PD1 antibody, the fusion protein comprising the polypeptides of SEQ ID NO: 15, 26, 16, and 4.

24. An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding one or more of the IL-12 variant, anti-PD1 antibody, fusion protein, or polypeptide thereof according to any one of claims 1 to 23.

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

45.

26. An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding VH, VL, or both of an antibody that binds to PD1, the polynucleotide comprising 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. **Claim 27** An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any one or more of the heavy chain, light chain, IL-12 p40 subunit, or heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD1 antibody, the polynucleotide comprising the heavy chain nucleic acid sequence of SEQ ID NO: 48, the light chain nucleic acid sequence of SEQ ID NO: 50, the IL-12 p40 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-12 p40 subunit nucleic acid sequence of SEQ ID NO: 45, and the heavy chain-IL12 p35 fusion polypeptide nucleic acid sequence of SEQ ID NO:

49. **Claim 28** An isolated polynucleotide or polynucleotides comprising one or more nucleotide sequences encoding any one or more of a heavy chain, a light chain, an IL-12 p40 subunit, or a heavy chain-IL12 p35 fusion polypeptide of a fusion protein comprising a human IL-12 variant and an anti-PD1 antibody, the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127517 for the heavy chain, the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127519 for the light chain, the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127520 for the IL-12 p40 subunit, the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127518 for the heavy chain-IL12 p35 fusion polypeptide, or the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127517 for the heavy chain, the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127519 for the light chain, the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127520 for the IL-12 p40 subunit, and the polynucleotide encoding the nucleic acid sequence of the insert of a plasmid deposited with the ATCC and having the ATCC accession number PTA-127518 for the heavy chain-IL12 p35 fusion polypeptide.

29. A vector comprising the polynucleotide or polynucleotides according to any one of claims 24 to 28.

30. An isolated host cell comprising the polynucleotide or polynucleotides according to any one of claims 24 to 28 or the vector according to 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 according to 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 an IL-12 variant, an anti-PD1 antibody, or a fusion protein according to any one of claims 1 to 23, 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 according to claim 32 or an IL-12 variant, an anti-PD1 antibody, or a fusion protein according to any one of claims 1 to 23.

34. An IL-12 variant, an anti-PD1 antibody, or a 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 and neck squamous cell carcinoma [squamous cell carcinoma of the head and neck (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 cancer, 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 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 different from the anti-PD1 antibody according to any one of claims 12 to 17.

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