INTERLEUKIN-21 MUTEINS AND TREATMENT METHODS

Abstract

The invention relates to IL-21 mutains and fusion proteins comprising them for use in methods of treating a disease. Conjugates, nucleic acids, vectors, host cells, pharmaceutical compositions, and associated kits are further described. The invention also relates to methods for preparing said IL-21 mutains and fusion proteins comprising them, as well as methods for treating a subject in need of them. Proteins binding to the pd-1 antigen are further described.

Description

BACKGROUND

[0001] The PD-1 / PD-L1 axis is involved in the suppression of T cell immune responses in cancer. Antagonists of this pathway have been clinically validated across a number of solid tumor indications. Nivolumab and pembrolizumab are two such inhibitors that target the PD-1 pathway, and each has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic melanoma. Recently, researchers have tested the paradigm of checkpoint inhibition in the setting of other tumor types. While some advances have been made, checkpoint inhibition therapy still remains in the shadows of other cancer treatment options.

[0002] Studies of checkpoint inhibitors in combination with other agents are underway or recently have been completed. The combination of nivolumab and ipilimumab, a CTLA-4 receptor blocking antibody, for example, was tested in a Phase III clinical trial on patients with unresectable stage III or IV melanoma. In this study, the percentage of patients achieving a complete response was the highest among those that received the combination of nivolumab and ipilimumab, beating the outcome exhibited by those in the group receiving either drug alone. Other combinations are also currently being explored.

[0003] Interleukin-21 (IL-21) is a T cell derived pleiotropic cytokine that regulates the activity of both innate and adaptive immune cells. IL-21 can augment T cell survival and effector function. Because it plays a key role in anti-tumor and anti-viral responses, in addition to exerting major effects on inflammatory responses that lead to the development of autoimmune diseases and inflammatory diseases, IL-21 has been an attractive target for several therapies.

[0004] However, the development of IL-21-based therapies has been complex. The research has been complicated by studies showing that enhancing, or confusingly, inhibiting IL-21 action leads to a therapeutic effect. Additional challenges exist due to the broad expression of the receptor for IL-21 (IL-21R). IL-21R is expressed not just on T cells, but also on B cells, NK cells and myeloid cells. Accordingly, care must be taken to limit broad IL-21 activation in leukocytes and avoid the potential for toxicity. Restriction of IL-21 signaling must be balanced and selective. Triggering the effects of IL-21 must be designed to occur at the right time and place.

[0005] Indeed, any success, especially clinical success with IL-21 moieties as a monotherapy or in combination with checkpoint inhibitors, has been muted. Thus, there remains a need for treatment modalities utilizing IL-21, including modalities combining IL-21 moieties with checkpoint inhibitors. There also remains a need for IL-21 therapies combined with immune checkpoint inhibition.SUMMARY OF THE INVENTION

[0006] The present invention provides a conjugate comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 2, wherein "X" represents any amino acid, and wherein said IL-21 mutein amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least 1 amino acid. The present invention further provides a kit comprising said conjugate and a container; a pharmaceutical composition comprising said conjugate and a pharmaceutically acceptable carrier, excipient, or diluent; and said pharmaceutical composition for use in treating a subject in need thereof. The invention is further defined in the appended claims.SUMMARY OF FURTHER DISCLOSURE

[0007] Described herein are IL-21 muteins comprising the amino acid sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 is QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), and X is any amino acid, and wherein the IL-21 mutein amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least 1 amino acid.

[0008] Thus, also described are IL-21 muteins comprising only one amino acid substitution, relative to the wild-type IL-21 amino acid sequence, which is provided herein as SEQ ID NO: 1. The amino acid substitution may be located at an amino acid position selected from the group consisting of: 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123, according to the amino acid position numbering of SEQ ID NO: 1.

[0009] Also described are IL-21 muteins comprising only two amino acid substitutions, relative to SEQ ID NO: 1. The amino acid substitutions may be located at two amino acid positions selected from the group consisting of: 5, 9, 15, 70, 71, 72, 73, and 76, according to the amino acid position numbering of SEQ ID NO: 1.

[0010] The IL-21 muteins may bind to the IL-21 receptor (IL-21R) with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 receptor. The IL-21 mutein may bind to the human IL-21R with a K D that is greater than or is about 0.04 nM. The IL-21 mutein may bind to the cynomolgus monkey IL-21R with a K D that is greater than or is about 0.055 nM.

[0011] Also described are conjugates comprising an IL-21 mutein as described herein linked to a heterologous moiety. The heterologous moiety may be a polypeptide, such that the conjugate is a fusion protein. Therefore, also described are fusion proteins comprising an IL-21 mutein as described herein. The fusion protein may comprise an IL-21 mutein of the present disclosure linked to an antigen-binding protein, such as an antibody, or an antigen binding antibody fragment thereof.

[0012] The fusion protein comprises an IL-21 mutein linked to a PD-1 antigen-binding antibody as described herein.

[0013] Also described are PD-1 antigen-binding proteins and conjugates and fusion proteins comprising a PD-1 antigen-binding protein.

[0014] Also described are nucleic acids comprising a nucleotide sequence encoding an IL-21 mutein, a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), or a fusion protein comprising an IL-21 mutein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody) as described herein. The nucleic acid molecule may comprise a nucleotide sequence encoding a conjugate or fusion protein as described herein. Vectors comprising the nucleic acids as described herein and host cells comprising the nucleic acids as described herein are furthermore described herein.

[0015] Also described are kits comprising an IL-21 mutein, a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), a conjugate, fusion protein (e.g., a fusion protein comprising an IL-21 mutein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody)), nucleic acid, vector, or host cell as described herein, or a combination thereof.

[0016] Pharmaceutical compositions comprising an IL-21 mutein, a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), a conjugate, fusion protein (e.g., a fusion protein comprising an IL-21 mutein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody)), nucleic acid, vector, or host cell as described herein, or a combination thereof, are also described herein.

[0017] Methods of making an IL-21 mutein, PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), and a fusion protein comprising an IL-21 mutein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody) are described herein. The method may comprise culturing a host cell as described herein to express the IL-21 mutein, PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), or a fusion protein comprising an IL-21 mutein and a PD-1 antigen-binding protein

[0018] (e.g., a PD-1 antigen-binding antibody) and harvesting the expressed IL-21 mutein, PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), or fusion protein comprising an IL-21 mutein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody).

[0019] Methods of treatment are additionally described herein. The method may be a method of treating a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition as described herein in an amount effective to treat the subject. The subject may have a tumor (e.g., a solid tumor, a hematological malignancy, or a lymphoid malignancy) and the pharmaceutical composition may be administered to the subject in an amount effective to treat the tumor in the subject. The tumor may be non-small cell lung cancer (NSCLC) (e.g., Stage III or IV NSCLC), small cell lung cancer (SCLC), head and neck cancer, renal cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, cancers with high microsatellite instability (i.e., MSI-high cancers), lymphoma or leukemia.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A represents a graph of the tumor volume (mm 3< ) of BALB / c mice implanted with CT26 / 3E5 colon carcinoma cells as a function of time (days). On Day 12, tumors were measured and the mice were given an intraperitoneal (IP) injection of 300 µg isotype control antibody (mIgG1) on day 12, 15 and 18. Figure 1B represents a graph of the tumor volume (mm 3< ) of BALB / c mice implanted with CT26 / 3E5 colon carcinoma cells as a function of time (days). On Day 12, tumors were measured and the mice were given an IP injection of 300 µg of an anti-PD-1 antibody on day 12, 15 and 18.. Figure 1C represents a graph of the tumor volume (mm 3< ) of BALB / c mice implanted with CT26 / 3E5 colon carcinoma cells as a function of time (days). On Day 12, tumors were measured and the mice were given 50 µg recombinant murine IL-21 (rmIL-21) three times per week for 3 weeks. Dosing ended on Day 33 Figure ID represents a graph of the tumor volume (mm 3< ) of BALB / c mice implanted with CT26 / 3E5 colon carcinoma cells as a function of time (days). On Day 12, tumors were measured and the mice were given 300 µg of an anti-PD-1 antibody on day 12, 15 and 18 and 50 µg rmIL-21 three times per week for 3 weeks. Dosing ended on Day 33. Figure 2 represents a graph of percent survival of the four groups of BALB / c mice implanted with CT26 / 3E5 colon carcinoma cells. Group 1 mice were given an intraperitoneal (IP) injection of 300 µg isotype control antibody (mIgG1) on day 12, 15 and 18. Group 2 mice were given an IP injection of 300 µg of an anti-PD-1 antibody on day 12, 15 and 18. Group 3 mice were given 50 µg rmIL-21 three times per week for 3 weeks. Group 4 mice were given 300 µg of an anti-PD-1 antibody on day 12, 15 and 18 and 50 µg rmIL-21 three times per week for 3 weeks. Administration of a combination of anti-PD-1 and rmIL-21 significantly extends survival as compared to monotherapy rmIL-21 or anti-PD-1. Figure 3 is an illustration of a hypothesis of the mechanism of action of a fusion protein comprising a blocking PD-1 antibody fused to an IL-21 mutein (αPD-1:IL-21 mutein). Without being bound by a particular theory, it is believed that the fusion binds to IL-21R on CD8+ T cells while simultaneously blocking the signal transduction between PD-1 and PD-L1. Figure 4A is an illustration of a fusion protein comprising an anti-PD-1 antibody fused to an IL-21 mutein homodimer. The fusion protein does not have a linker. The antibody may comprise constant regions that reduce or eliminate Fc-associated effector binding and functions (e.g., lack the ability to interact with Fcγ receptors (e.g., SEFL2-2)). Figure 4B is an illustration of a fusion protein comprising an anti-PD-1 antibody fused to an IL-21 mutein homodimer. The fusion protein may comprise a GGGGS (G4S) linker between the heavy chain constant region of the antibody and the IL-21 mutein. The antibody may also comprise SEFL2-2 modifications. Figure 4C is an illustration of a fusion protein comprising an anti-PD-1 antibody fused to an IL-21 mutein monomer. The fusion protein may comprise a G4S linker between the heavy chain constant region of the antibody and the IL-21 mutein. The antibody heavy chains comprise charge pair mutations (cpm; e.g., V1, V4, V103, or V131) to aid in preferential association of heterodimer Fc regions. The antibody may also comprise SEFL2-2 modifications. Figure 5A represents a graph of the STAT3 signaling in PD-1 -ve< Hut78 T cells exposed to (i) recombinant human IL-21 (rhIL-21) alone (solid line with closed circles), (ii) anti-PD-1 mAb alone (solid line with closed diamonds), (iii) anti-PD-1 mAb fused to an IL-21 homodimer without linker (solid line with closed triangles), (iv) anti-PD-1 mAb fused to an IL-21 homodimer with linker (dashed line with open triangles), (v) anti-PD-1 mAb fused to an IL-21 monomer without linker (solid line with closed squares), or (vi) anti-PD-1 mAb fused to an IL-21 monomer with a linker (dashed line with open squares). Figure 5B represents a graph of the STAT3 signaling in PD-1 +ve< Hut78 T cells exposed to (i) recombinant human IL-21 (rhIL-21) alone (solid line with closed circles), (ii) anti-PD-1 mAb alone (solid line with closed diamonds), (iii) anti-PD-1 mAb fused to an IL-21 homodimer without linker (solid line with closed triangles), (iv) anti-PD-1 mAb fused to an IL-21 homodimer with a linker (dashed line with open triangles), (v) anti-PD-1 mAb fused to an IL-21 monomer without a linker (solid line with closed squares), or (vi) anti-PD-1 mAb fused to an IL-21 monomer with a linker (dashed line with open squares). Figure 6 represents a graph of the serum concentrations of a homodimer fusion protein comprising WT IL-21 fused to an anti-PD-1 mAb that was intravenously administered to 6 animals at either a low dose (250 µg / kg) or a high dose (1000 µg / kg). An IgG antibody domain (150 µg / kg) was run as a control. Figure 7 represents a graph of the fold reduction in IL-21 activity (relative to rhIL-21 activity) by PD-1 -ve< Hut78 cells (open bars) or PD-1 +ve< Hut78 cells (closed bars) exposed to IL-21 muteins with reduced affinity for IL-21Rα. Figure 8 represents a graph of the fold reduction in IL-21 activity (relative to rhIL-21 activity) by PD-1 -ve< Hut78 cells (open bars) or PD-1 +ve< Hut78 cells (closed bars) exposed to IL-21 muteins with reduced affinity for IL-21Rγ. Figure 9A represents a graph of the STAT3 signaling in PD-1 -ve< Hut78 T cells exposed to (i) recombinant human IL-21 (rhIL-21) alone (solid line with closed circles), (ii) anti-PD-1 mAb fused to a WT IL-21 homodimer (dashed line with open circles), (iii) anti-PD-1 mAb fused to a WT IL-21 monomer (dashed line with closed circles), and (iv) anti-PD-1 mAb fused to an IL-21 mutein 51 homodimer (R65P) (dotted line with closed triangles). Figure 9B represents a graph of the STAT3 signaling in PD-1 +ve< Hut78 T cells exposed to (i) recombinant human IL-21 (rhIL-21) alone (solid line with closed circles), (ii) anti-PD-1 mAb fused to a WT IL-21 homodimer (dashed line with open circles), (iii) anti-PD-1 mAb fused to a WT IL-21 monomer (dashed line with closed circles), and (iv) anti-PD-1 mAb fused to an IL-21 mutein 51 homodimer (R65P) (dotted line with closed triangles). Figure 10 represents a graph of the serum concentrations of a fusion protein comprising (i) an anti-PD-1 mAb fused to an IL-21 R76E mutein homodimer (dotted line with closed circles), (ii) an anti-PD-1 mAb fused to an IL-21 R76A mutein homodimer (dashed line with closed triangles), (iii) an anti-PD-1 mAb fused to an IL-21 D15N mutein homodimer (dashed line with X's), (iv) an anti-PD-1 antibody (8.25 mg / kg; dashed line with open diamonds), and (v) an anti-PD-1 mAb fused to a WT IL-21 mutein homodimer (solid line with closed squares). Figure 11 represents a graph of the IL-2 (pg / mL) secreted by cells of a mixed lymphocyte reaction as a function of antibody concentration of (i) an anti-PD-1 antibody (solid line with closed circles), (ii) a fusion protein comprising IL-21 R5Q / R76E double mutein homodimer (dashed line with open circles), (iii) a combination of anti-PD-1 mAb and rhIL-21 (dotted line with open squares), (iv) a fusion protein comprising IL-21 R76E single mutein homodimer (closed squares), (v) IgG control (dotted line with open diamonds), and (vi) a rhIL-21 (broken line with open triangles). Figure 12A represents a graph of the fold change of over mean fluorescence intensity (MFI) of STAT3 activity of cells exposed to (i) a fusion protein comprising an IL-21 R5E / R76A double mutein (line with open triangles), (ii) an IgG1 control (dotted line with closed diamonds), (iii) rhIL-21 (dashed line with open squares), (iv) an anti-PD-1 mAb (solid line with open ovals), (v) a combination of rhIL-21 and anti-PD-1 mAb (dashed line with closed ovals), or (vi) a fusion protein comprising an IL-21 R76E single mutein (line with open diamonds). Figure 12B represents a graph of the fold change of over MFI of STAT3 activity of cells exposed to (i) a fusion protein comprising an IL-21 R5Q / R76E double mutein (line with open triangles), (ii) an IgG1 control (dotted line with closed diamonds), (iii) rhIL-21 (dashed line with open squares), (iv) an anti-PD-1 mAb (solid line with open ovals), (v) a combination of rhIL-21 and anti-PD-1 mAb (dashed line with closed ovals), or (vi) a fusion protein comprising an IL-21 R76E single mutein (line with open diamonds). Figure 12C represents a graph of the fold change of over MFI of STAT3 activity of cells exposed to (i) a fusion protein comprising an IL-21 R9E / R76A double mutein (line with open triangles), (ii) an IgG1 control (dotted line with closed diamonds), (iii) rhIL-21 (dashed line with open squares), (iv) an anti-PD-1 mAb (solid line with open ovals), (v) a combination of rhIL-21 and anti-PD-1 mAb (dashed line with closed ovals), or (vi) a fusion protein comprising an IL-21 R76E single mutein (line with open diamonds). Figure 13A represents a graph of the % specific lysis by CTLs (y-axis) vs effector to target cell ratios (x-axis) exposed to (i) rhIL-21 (solid line with closed circles), (ii) hIgG4 control antibody (dashed line with open circles), (iii) anti-PD-1 mAb (dashed line with closed triangles), (iv) a combination of rhIL-21 and anti-PD-1 mAb (dotted line with open triangles), or (v) a fusion protein comprising IL-21 R5E / R76A (broken line with X's). Figure 13B represents a graph of the % specific lysis by CTLs exposed to represents a graph of the % specific lysis by CTLs exposed to (i) rhIL-21 (solid line with closed circles), (ii) hIgG4 control antibody (dashed line with open circles), (iii) anti-PD-1 mAb (dashed line with closed triangles), (iv) a combination of rhIL-21 and anti-PD-1 mAb (dotted line with open triangles), or (v) a fusion protein comprising IL-21 R5Q / R76E (broken line with X's). Figure 13C represents a graph of the % specific lysis by CTLs exposed to (i) rhIL-21 (solid line with closed circles), (ii) hIgG4 control antibody (dahsed line with open circles), (iii) anti-PD-1 mAb (dashed line with closed triangles), (iv) a combination of rhIL-21 and anti-PD-1 mAb (dotted line with open triangles), or (v) a fusion protein comprising IL-21 R9E / R76A (broken line with X's). Figure 14 represents serum concentrations of (i) anti-PD-1 mAb (solid line with closed squares), (ii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R5Q / R76E homodimer (solid line with closed circles), (iii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E homodimer (solid line), (iv) a fusion protein comprising anti-PD-1 mAb and an IL-21 R5E / R76A homodimer (dashed line with X's), (v) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76A homodimer (dashed line with closed diamonds), or (vi) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E monomer (solid line with closed triangles). Figure 15A represents a timeline of the cell sampling (open arrows) and of the administrations (closed arrows). Figure 15B represents a graph of the fold change in PD-1 +< / CD4 +< cells (relative to Day -5) as measured on Day 7 in animals given a dose of (i) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E mutein (bar with horizontal lines), (ii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E single mutein monomer (bar with vertical lines), or (iii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R5Q / R76E double mutein homodimer (open bar) (each administered on Day 0). Figure 15C represents a graph of the fold change in PD-1 +< / CD8 +< cells (relative to Day -5) as measured on Day 7 in animals given a dose of (i) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E mutein (bar with horizontal lines), (ii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E single mutein monomer (bar with vertical lines), or (iii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R5Q / R76E double mutein homodimer (open bar) (each administered on Day 0). Figure 15D represents a graph of the fold change in PD-1 +< / CD8 +< cells (relative to Day -5) as measured on Day 21 in animals given a first dose of (i) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E mutein (bar with horizontal lines), (ii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R76E single mutein monomer (bar with vertical lines), or (iii) a fusion protein comprising anti-PD-1 mAb and an IL-21 R5Q / R76E double mutein homodimer (open bar) (first dose administered on Day 0 and second dose administered on Day 8). Figure 16 represents a graph of the expansion of PD-1 +< / CD8 +< cells (relative to Day -5) as a function of PD-1 receptor occupancy (RO) on PD-1 +< / CD8 +< cells. IL-21 single mutants (801, 802, 807, and 808) are shown within the blue circle and IL-21 double mutants (803, 804, 805, and 806) are shown in the pink circle. These data demonstrate that superior pharmacokinetic properties of the double mutants allow for better target coverage and correlates with better pharmacodynamics responses, as measured by expansion of the PD-1+ target population. The blue line indicates the general delineation between single and double mutants. Figure 17 represents flow plots of individual animals treated with either single mutant homodimer constructs (animals 801 and 802), double mutant homodimer constructs (animals 803 - 806), or single mutant monomeric constructs (animals 807 and 808). PD-1+ T cells were detected by non-competing or competing detection antibody. Percent target coverage is calculated as a percentage of the ratio between competing and non-competing PD-1 antibody. The data show that all constructs demonstrate robust target coverage after repeat dosing on Day 21. Figure 18A represents a graph of the fold change (relative to rhIL-21 (solid line with circled X's)) of STAT3 activity of PD-1 -ve< Hut78 T cells exposed to a fusion protein comprising one of ten different anti-PD-1 mAbs and an IL-21 R5Q / R76E mutein homodimer. The ten anti-PD-1 mAbs included 20A2.003 (line with diamonds), 20C1.006 (red square), 20C1.009 (line with triangles), and 22D4.006 (line with open circles). Figure 18B represents a graph of the fold change (relative to rhIL-21 (solid line with circled X's)) of STAT3 activity of PD-1+ve Hut78 T cells exposed to a fusion protein comprising one of ten different anti-PD-1 mAbs and an IL-21 R5Q / R76E mutein homodimer. The ten anti-PD-1 mAbs included 20A2.003 (line with diamonds), 20C1.006 (red square), 20C1.009 (line with triangles), and 22D4.006 (line with open circles). Comparison of Figure 18A to Figure 18B indicates that PD-1 targeting needed for pSTAT3 signaling and that the activity of the muteins is similar when fused to different anti-PD-1 mAbs. Figure 19A represents a graph of the fold change (relative to rhIL-21 (solid line with circled X's)) of STAT3 activity of PD-1 -ve< Hut78 T cells exposed to a fusion protein comprising one of seven different anti-PD-1 mAbs and an IL-21 R9E / R76A mutein homodimer. The seven anti-PD-1 mAbs included 20A2.003 (line with open triangles), 20C1.006 (line with open squares), 20C1.009 (line with open diamonds), and 22D4.006 (line with open circles). Figure 19B represents a graph of the fold change (relative to rhIL-21 (solid line with circled X's)) of STAT3 activity of PD-1 +ve< Hut78 T cells exposed to a fusion protein comprising one of seven different anti-PD-1 mAbs and an IL-21 R9E / R76A mutein homodimer. Comparison of Figure 19A to Figure 19B indicates that PD-1 targeting needed for pSTAT3 signaling and that the activity of the muteins is similar when fused to different anti-PD-1 mAbs. The seven anti-PD-1 mAbs included 20A2.003 (line with open triangles), 20C1.006 (line with open squares), 20C1.009 (line with open diamonds), and 22D4.006 (line with open circles). Figures 20A-20D represent the amount of pSTAT3 signaling observed with several anti-PD-1 mAb - IL-21 monomeric or dimeric double mutein fusions. Solid line with closed circles(top of graphs) is rhIL-21; dashed line with open circles (bottom of graphs) is IgG1 control; line with X's (bottom of graphs) is IgG2 control; dotted line with closed squares (bottom of graphs) is the 22D4.006 anti-PD-1 mAb (present as mAb; i.e., not as a fusion) used in the IL-21 mutein fusions; dashed line with open squares and dotted line with open diamonds(bottom of graphs) are control anti-PD-1 mAbs; remaining lines are anti-PD-1 mAb (22D4.006) - IL-21 monomeric or dimeric double mutein fusions (with various charge pair mutations) wherein the double mutants are R5E / R76A; R9E / R76A; R5A / R76E or R5Q / R76E. rhIL-21 demonstrates activity in both PD-1 -ve< and PD-1 +ve< cells, monomeric and homodimeric double mutein fusions are unable to demonstrate pSTAT3 (IL-21-based) activity in PD-1 -ve< cells, and monomeric and homodimeric double mutein fusions are able to demonstrate pSTAT3 (IL-21-based) activity in PD-1 +ve< cells. Thus, monomeric fusions with IL-21 double mutants exhibit similar levels of IL-21 activity attenuation in PD-1 -ve< cells and IL-21 activity rescue in PD-1 +ve< cells as their counterpart dimeric fusions. Figures 20A and 20B are duplicate runs of the pSTAT3 assay on PD-1 -ve< cells. Figures 20C and 20D are duplicate runs of the pSTAT3 assay on PD-1 +ve< cells. Figures 21A-21D represent the results of a PD-1 reporter gene assay (RGA; Figures 21A and 21B) and MLR assay (Figures 21C and 21D) with the same anti-PD-1 mAb (22D4.006) - IL-21 monomeric or dimeric double mutein fusions evaluated in Figures 20A-20D. Figures 21A-21D demonstrate that anti-PD-1 mAb (22D4.006) - IL-21 monomeric and dimeric double mutein fusions are able to induce PD-1 activity. Solid line with closed circles (bottom of graphs) is rhIL-21; line with open circles (bottom of graphs) is IgG1 control; line with X's (bottom of graphs) is IgG2 control; dashed line with closed squares (top of graphs) is the 22D4.006 anti-PD-1 mAb (present as mAb; i.e., not as a fusion) used in the IL-21 mutein fusions; dashed line with open squares and dotted line with open diamonds (top of graphs) are control anti-PD-1 mAbs; remaining lines are anti-PD-1 mAb - IL-21 monomeric or dimeric double mutein fusions. Figures 21A and 21B are duplicate runs of the PD-1 RGA assay. Figures 20C and 20D are duplicate runs of the MLR assay. Figures 22A-22D represent the results of pSTAT3 assays testing the same constructs as those in Figures 20A-20D, except that a different anti-PD-1 mAb (20A2.003) is used in the anti-PD-1 mAb - IL-21 monomeric and dimeric double mutein fusions. The results in Figures 22A-22D are similar to those seen in Figures 20A-20D. Solid line with closed circles (top of graphs) is rhIL-21; dashed line with open circles (bottom of graphs) is IgG1 control; line with X's (bottom of graphs) is IgG2 control; dotted line with closed squares (bottom of graphs) is the 20A2.003 anti-PD-1 mAb (present as mAb; i.e., not as a fusion) used in the IL-21 mutein fusions; dashed line with open squares and dotted line with open diamonds (bottom of graphs) are control anti-PD-1 mAbs; remaining lines are anti-PD-1 mAb (20A2.003) - IL-21 monomeric or dimeric double mutein fusions (with various charge pair mutations) wherein the double mutants are R5E / R76A; R9E / R76A; R5A / R76E or R5Q / R76E. Figures 22A and 22B are duplicate runs of the pSTAT3 assay on PD-1 -ve< cells. Figures 22C and 22D are duplicate runs of the pSTAT3 assay on PD-1 +ve< cells. Figures 23A-23D represent the results of PD-1 reporter gene assays (Figures 23A and 23B) and MLR assays (Figures 23C and 23D) testing the same constructs as those in Figures 21A-21D, except that a different anti-PD-1 mAb (20A2.003) is used in the anti-PD-1 mAb - IL-21 monomeric and dimeric double mutein fusions. The results in Figures 23A-23D are similar to those seen in Figures 21A-21D. Solid line with closed circles (bottom of graphs) is rhIL-21; dashedline with open circles (bottom of graphs) is IgG1 control; line with X's (bottom of graphs) is IgG2 control; dotted line (top of graphs) is the 20A2.003 anti-PD-1 mAb (present as mAb; i.e., not as a fusion) used in the IL-21 mutein fusions; dashed line with open squares and dotted line with open diamonds (top of graphs) are control anti-PD-1 mAbs; remaining lines are anti-PD-1 mAb (20A2.003) - IL-21 monomeric or dimeric double mutein fusions. Figures 23A and 23B are duplicate runs of the PD-1 RGA assay. Figures 23C and 23D are duplicate runs of the MLR assay. Figure 24 represents a graph of the NFAT / luciferase activity of purified anti-PD-1 antibodies as a function of mAb concentration. Figures 25A is a graph of the fold change in the number of Ki67+ / CD3+ / CD4+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25B is a graph of the fold change in the number of Ki67+ / CD3+ / CD8+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25C is a graph of the fold change in the number of pSTAT3+ / CD3+ / CD4+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25D is a graph of the fold change in the number of pSTAT3+ / CD3+ / CD8+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25E is a graph of the fold change in the number of CD3+ / CD4+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25F is a graph of the fold change in the number of CD3+ / CD8+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25G is a graph of the fold change in the number of PD-1+ / CD3+ / CD4+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25H is a graph of the fold change in the number of PD-1+ / CD3+ / C84+ cells relative to baseline upon exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25I is a graph of the fold change in the amount of serum perforin relative to baseline upon 72 hours of exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figures 25J is a graph of the % Ki67+ cells relative to baseline as a function of fold increase in perforin upon 72 hour exposure to the fusion protein [22D4.017]-[R9E:R76A] (monomer) or to the anti-PD-1 antibody [22D4.017]. Figure 26A is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of antibody 22D4.017 for human PD-1 antigen. Figure 26B is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of antibody 20C 1.009 for human PD-1 antigen. Figure 26C is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of antibody 20A2.003 for human PD-1 antigen. Figure 26D is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of an IgG1 anti-PD-1 mAb, for human PD-1 antigen. Figure 26E is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of an IgG4 PD-1 mAb for human PD-1 antigen. Figure 26F is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of 22D4.017 for cyno PD-1 antigen. Figure 26G is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of antibody 20C1.009 for cyno PD-1 antigen. Figure 26H is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of antibody20A2.003 for cyno PD-1 antigen. Figure 26I is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of an IgG1 anti-PD-1 mAb for cyno PD-1 antigen. Figure 26J is a graph of the absorbance (nm) as a function of time (sec) used to determine the indicated K D of an IgG4 anti-PD-1 mAb for cyno PD-1 antigen. Figure 27 is a graph of the Cp (kcal / mole / ° C) as a function of temperature for anti-PD-1 antibodies 22D4.017 and 20C1.009. Figure 28 is a graph of the viscosity plotted against shear rate for anti-PD-1 antibodies 22D4.017 and 20C1.009. Figures 29A-29D is a series of graphs plotting the signal as a function of antibody concentration in (Fig. 29A) a variant Hut78 T cell line that is PD-1 positive, (Fig. 29B) a variant Hut78 T cell line that is TIGIT positive, (Fig. 29C) a variant Hut78 T cell line that is LAG3 positive, and (Fig. 29D) the parental Hut78 T cell line which does not endogenously express PD-1, TIGIT or LAG3. DETAILED DESCRIPTION

[0021] There remains a need for novel immune potentiating approaches that can deploy the immune system against cancer cells in a safe and efficacious manner, especially in light of the fact that current immunotherapy approaches are efficacious in only a minority of patients, and can have significant and often unpredictable toxicities. Described herein is a novel class of bifunctional fusion molecules comprising a PD-1 targeting antibody that can block PD-1 / PD-L1 interaction, fused to an engineered affinity attenuated interleukin-21 mutein which addresses this need. The antibody / cytokine fusions described herein overcome significant barriers associated with cytokine therapeutics, allowing for, inter alia, antibody-like dosing and selective delivery of the IL-21 cytokine in a PD-1 targeted manner. When fused to an anti-PD-1 antibody, IL-21 muteins can selectively activate and expand PD-1 expressing T cells in vivo. Accordingly, the antibody / cytokine fusions described herein may improve upon and extend the utility of anti-PD-1 therapeutics currently under testing in the clinic.

[0022] The combination of cytokine and co-inhibitory receptor agonists or antagonists remains challenging because of the risks of incremental toxicity and the need for complex clinical trial design (see, e.g., Ott et al., J Immunother Cancer 5, 16 (2017); and Hermel et al., Cancer Metastasis Rev 36, 43-50 (2017)). With respect to cytokines, there is also the potential for the activation of inhibitory feedback pathways that can lead to immune suppression (see, e.g., Portielje et al, Clin Cancer Res 9, 76-83 (2003); Wan et al., Immunity 38, 514-527 (2013); and Mooradian et al., Oncoimmunology 7, e1423172 (2018)). Interleukin-21 (IL-21) is a type I cytokine and a member of the common cytokine receptor gamma-chain (cg-chain) cytokine family that has emerged as a promising immune therapeutic for the treatment of cancer. IL-21 is produced by activated CD4 T cells and natural killer T (NKT) cells and signals via a heterodimeric receptor complex comprised of a discrete IL-21 receptor (IL-21R) subunit that associates with the common gamma-chain (see, e.g., Spolski et al., Nat Rev Drug Discov 13, 379-395 (2014)). Activation of the IL-21R complex leads to the activation of the JAK / STAT signaling pathway. IL-21R is broadly expressed in hematopoietic cells including T and B lymphocytes, natural killer (NK) cells and myeloid cells. Although not an essential growth or differentiation factor, IL-21 is a potent mitogen and survival factor for NK cells and activated T cells. IL-21 can support the differentiation of CD4 (+) T helper 17 (Th17) as well follicular helper T cells (Tfh) and can antagonize regulatory T cell (Treg) differentiation. Moreover, IL-21 can augment the survival of CD8 T cells and preserves a less activated but more persistent T cell phenotype, which allows for enhanced tumor and viral control.

[0023] A challenging aspect of cytokine immunotherapy is that, in addition to activating immune cells to potentiate immune responses, the same cytokine can also activate counter-regulatory pathways. For example, IL-2 and IFNγ which can activate protective immune responses as well as regulatory T cell responses and inhibitory pathways (such as PD-L1) respectively. In dendritic cells (DCs), IL-21 can inhibit both DC maturation and activation, can induce the apoptosis of conventional DCs, can potently inhibit the priming of T cells in mixed cultures, and may play a role in the induction of tolerance. In humans, IL-21 has been tested as a non-targeted free cytokine in several cancer indications, but despite the promising preclinical data and early phase I clinical data, development of this approach has not progressed further than phase II testing (see, e.g., Thompson et al., J Clin Oncol 26, 2034-2039 (2008); and Davis et al., Clin Cancer Res 15, 2123-2129 (2009)). In more recent preclinical models, the combination of recombinant IL-21 cytokine with co-inhibitory receptor antagonists (e.g., anti-CTLA-4 and anti-PD-1) have demonstrated that IL-21 can extend the efficacy of these treatments. Such combinations are now under testing in the clinic, though clinical efficacy has yet to be demonstrated (Lewis et al., Oncoimmunology 7, e1377873 (2017)).

[0024] Without being bound by a theory, the antibody / cytokine fusions described herein are designed to utilize the immune potentiating activity of IL-21 (which may be prerequisite to address toxicity and off-target immune suppression), to maximize efficacy, and improve the feasibility of dosing in the clinic. IL-21 and IL-21 muteins

[0025] Interleukin-21 (IL-21) is a cytokine expressed by T cells, B cells, NK cells and myeloid cells, and regulates the activity of both innate and adaptive immune cells and improves T cell survival and effector function. Several Phase I and II clinical trials include IL-21 as the investigational product for the treatment of cancers, inflammatory diseases, and autoimmune diseases, including, melanoma, renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, ovarian cancer, colorectal cancer, systemic lupus erythematosus, Crohn's disease and rheumatoid arthritis.

[0026] IL-21 has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each of which are derived from a precursor molecule. The first IL-21 isoform comprises 162 amino acids (aa), the first 29 of which make up the signal peptide; and the second IL-21 isoform comprises 153 aa, the first 29 of which make up the signal peptide as in the first isoform. The amino acid sequences of the first and second IL-21 isoforms (including the signal peptide) are provided herein as SEQ ID NO: 258 and SEQ ID NO: 259, respectively.

[0027] IL-21 binds to the heterodimeric IL-21 receptor (IL-21R) expressed on the surface of T, B, and NK cells. IL-21R is similar in structure to the IL-2 receptor and the IL-15 receptor, in that each of these cytokine receptors comprises a common gamma chain (γc). In addition to the γc, the IL-21R comprises an alpha chain which is important for binding to IL-21. There are two isoforms of the human IL-21 receptor alpha chain: isoform 1 and isoform 2. The amino acid sequences of isoform 1 and isoform 2 are provided herein as SEQ ID NOs: 256 and 261, respectively. The amino acid sequence of the human common gamma chain is provided herein as SEQ ID NO: 257.

[0028] When IL-21 binds to IL-21R, the Jak / STAT signaling pathway is activated to activate target genes. While IL-21-induced signaling may be therapeutically desirable, careful consideration of the timing and the location of the signaling is needed, given IL-21's broad expression profile and due to the fact that IL-21 has the ability to potentiate CD8 T cell responses as well as to suppress antigen presentation and T cell priming. The data presented herein for the first time supports the use of carefully designed IL-21 muteins to achieve IL-21 signaling at the appropriate time and place.

[0029] Described herein are IL-21 muteins comprising at least one amino acid substitution, relative to the wild-type IL-21 amino acid sequence, which is provided herein as SEQ ID NO: 1. For example, the IL-21 mutein comprises at least one and not more than 34 amino acid substitutions. The IL-21 mutein may comprise at least one and not more than X amino acid substitutions, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 7 amino acids or no more than 5 amino acids. The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5, or 6 amino acids. The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3 to 6 amino acids or 1 to 5 amino acids. The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by one or two amino acids.

[0030] The IL-21 mutein may comprise the amino acid sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 is QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), wherein X represents any amino acid, and wherein the IL-21 mutein amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least 1 amino acid.

[0031] Thus, the IL-21 mutein may comprise the sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 differs from SEQ ID NO: 1 by at least one amino acid at a position designated by X in SEQ ID NO: 2. The IL-21 mutein comprising SEQ ID NO: 2 may have at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1. The IL-21 mutein may comprise an amino acid sequence which is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1.

[0032] The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution(s) may occur within the N-terminal half of the amino acid sequence. For example, the amino acid substitution(s) occur(s) at a position within positions 5-25 or 8-23 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 1.

[0033] The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution(s) may occur within the C-terminal half of the amino acid sequence. For example, the amino acid substitution(s) occur(s) at a position within positions 100-133 or 109-123 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 1.

[0034] The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution(s) may occur in the middle third of the amino acid sequence. For example, the amino acid substitution(s) occur(s) at a position within positions 55-85 or 65-80 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 1.

[0035] Also described are IL-21 muteins comprising only one amino acid substitution, relative to the wild-type IL-21 amino acid sequence, which is provided herein as SEQ ID NO: 1. The amino acid substitution may be located at an amino acid position selected from the group consisting of: 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123, according to the amino acid position numbering of SEQ ID NO: 1. The amino acid substitution may be located at an amino acid position selected from the group consisting of: 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123, according to the amino acid position numbering of SEQ ID NO: 1. The IL-21 mutein may comprise any one of the amino acid sequences of SEQ ID NOs: 3-21 and 23-37.

[0036] Also described are IL-21 muteins comprising only two amino acid substitutions, relative to SEQ ID NO: 1. The amino acid substitutions may be located at two amino acid positions selected from the group consisting of: 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123, according to the amino acid position numbering of SEQ ID NO: 1. The amino acid substitutions may be located at two amino acid positions selected from the group consisting of: 5, 9, 15, 70, 71, 72, 73, and 76, according to the amino acid position numbering of SEQ ID NO: 1. The amino acid substitutions may be located at two amino acid positions selected from the group consisting of: 5, 9, 73, and 76, according to the amino acid position numbering of SEQ ID NO: 1. At least one of the two amino acid substitutions may be located at position 76, according to the amino acid position numbering of SEQ ID NO: 1. The IL-21 mutein may comprise any one of the amino acid sequences of SEQ ID NOs: 199-208 and 210-212.

[0037] The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution(s) may be conservative amino acid substitution(s). As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues : Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their amides and esters : Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Polar, positively charged residues : His, Arg, Lys; Ornithine (Orn) IV. Large, aliphatic, nonpolar residues : Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine V. Large, aromatic residues : Phe, Tyr, Trp, acetyl phenylalanine.

[0038] The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution(s) may be non-conservative amino acid substitution(s). As used herein, the term "non-conservative amino acid substitution" is defined herein as the substitution of one amino acid with another amino acid having different properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges outside the above five groups.

[0039] The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the substitute amino acid may be a naturally-occurring amino acid. By "naturally-occurring amino acid" or "standard amino acid" or "canonical amino acid" is meant one of the 20 alpha amino acids found in eukaryotes encoded directly by the codons of the universal genetic code (Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Arg, His, Lys, Asp, Glu). The IL-21 mutein may comprise an amino acid sequence comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the substitute amino acid may be a non-standard amino acid, or an amino acid which is not incorporated into proteins during translation. Non-standard amino acids include, but are not limited to: selenocysteine, pyrrolysine, ornithine, norleucine, β-amino acids (e.g., β-alanine, β-aminoisobutyric acid, β-phenlyalanine, β-homophenylalanine, β-glutamic acid, β-glutamine, β-homotryptophan, β-leucine, β-lysine), homo-amino acids (e.g., homophenylalanine, homoserine, homoarginine, monocysteine, homocystine), N-methyl amino acids (e.g., L-abrine, N-methyl-alanine, N-methyl-isoleucine, N-methyl-leucine), 2-aminocaprylic acid, 7-aminocephalosporanic acid, 4-aminocinnamic acid, alpha-aminocyclohexanepropionic acid, amino-(4-hydroxyphenyl)acetic acid, 4-amino-nicotinic acid, 3-aminophenylacetic acid, and the like.

[0040] The IL-21 mutein of the present disclosure may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, and 119 of SEQ ID NO: 1, and the substitute amino acid(s) may be aliphatic amino acids. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid may be an aliphatic amino acid.

[0041] The IL-21 mutein of the present disclosure may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, and the substitute amino acid(s) may be acidic amino acids. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, and the substitute amino acid may be an acidic amino acid.

[0042] The IL-21 mutein as described herein may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1, and the substitute amino acid(s) may be basic amino acids. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, and the amino acid at position 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1 may be a basic amino acid.

[0043] The IL-21 mutein as described herein may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 5, 8, 9, 70, or 76 of SEQ ID NO: 1, and the substitute amino acid(s) may be aromatic amino acids. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 5, 8, 9, 70, or 76 of SEQ ID NO: 1, and the substitute amino acid may be an aromatic amino acid.

[0044] The IL-21 mutein as described herein may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid(s) may be amino acids comprising a side chain amide. The IL-21 mutein as described herein comprises an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid may be an amino acid comprising a side chain amide.

[0045] The IL-21 mutein as described herein may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid(s) may be amino acids comprising a side chain hydroxyl. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid may be an amino acid comprising a side chain hydroxyl.

[0046] The IL-21 mutein as described herein may comprise an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution may be at one or more of positions 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, and the substitute amino acid(s) may be imino acids. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, and the substitute amino acid may be an amino acid comprising an imino acid.

[0047] The IL-21 mutein as described herein comprises an amino acid sequence with at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), the amino acid substitution at one or more of positions 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid(s) may be amino acids comprising a sulfur-containing side chain. The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, the amino acid substitution may be at position 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, and the substitute amino acid may be an amino acid comprising an amino acid comprising a sulfur-containing side chain.

[0048] The IL-21 mutein as described herein may comprise an amino acid sequence with at least one amino acid substitution, relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the at least one amino acid substitution is shown in Table A. TABLE AAmino Acid position of SEQ ID NO: 1Examples of Substitute Amino Acids (in single letter code)Exemplary SEQ ID NO:5A, D, E, G, H, I, K, L, M, N, Q, S, T, V, Y388A, D, E, G, N, S399A, D, E, G, H, I, K, L, M, N, Q, S, T, V, Y4011D, S4112A, D, E, N, S, T, V4213D4314A, D, S4415A, E, I, M, N, Q, S, T, V4516D, E4619D4723D4865D, G, P4966D, G, P5068Q5169D, G, P5270E, G, P, Y, T5371L5472A, D, G, P5573A, D, E, G, H, I, N, P, Q, S, V5675D, G, P5876A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V, Y5977D, G, P6078D6179D6280G, P63109K64110D65112D66113K67116A, D, E, I, K, L, M, N, S, T, V68117D69119A, D, E, M, N, Q, S, T70120D71123D72

[0049] The IL-21 mutein as described herein may comprise an amino acid sequence with only one amino acid substitution, relative to SEQ ID NO: 1, and the amino acid substitution may be one shown in Table A. The IL-21 mutein as described herein may comprise an amino acid sequence with two amino acid substitutions, relative to SEQ ID NO: 1, and the amino acid substitutions may be two of those shown in Table A.

[0050] The IL-21 mutein as described herein may comprise an amino acid sequence shown in Table B. TABLE BAmino Acid SubstitutionAmino Acid position of SEQ ID NO: 1Substitute Amino AcidSEQ ID NO:Amino Acid SubstitutionAmino Acid position of SEQ ID NO: 1Substitute Amino AcidSEQ ID NO:R5A5A73S70E70E136R5D5D74S70G70G137R5E5E75S70P70P138R5G5G76S70Y70Y139R5H5H77S70T70T140R5I5I78K72D72D141R5K5K79K72G72G142R5L5L80K72P72P143R5M5M81K72A72A144R5N5N82K73A73A145R5Q5Q83K73D73D146R5S5S84K73E73E147R5T5T85K73G73G148R5V5V86K73H73H149R5Y5Y87K73I73I150I8A8A88K73N73N151I8D8D89K73P73P152I8E8E90K73Q73Q153I8G8G91K73S73S154I8N8N92K73V73V155I8S8S93K75D75D156R9A9A94K75G75G157R9D9D95K75P75P158R9E9E96R76A76A159R9G9G97R76D76D160R9H9H98R76E76E161R9I9I99R76G76G162R9K9K100R76H76H163R9L9L101R76I76I164R9M9M102R76K76K165R9N9N103R76L76L166R9Q9Q104R76M76M167R9S9S105R76N76N168R9T9T106R76P76P169R9V9V107R76Q76Q170R9Y9Y108R76S76S171R11D11D109R76T76T172R11S11S110R76V76V173Q12A12A111R76Y76Y174Q12D12D249K77D77D175Q12E12E250K77G77G176Q12N12N251K77P77P177Q12S12S252P78D78D61Q12T12T253P79D79D62Q12V12V254S80G80G178L13D13D112S80P80P179I14A14A114E109K109K64I14D14D115R110D110D65I14S14S116K112D112D66D15A15A117S113K113K67D15E15E118Q116A116A180D15I15I119Q116D116D181D15M15M120Q116E116E182D15N15N121Q116I116I183D15Q15Q122Q116K116K184D15S15S123Q116L116L185D15T15T283Q116M116M186D15V15V124Q116N116N187I16D16D125Q116S116S188I16E16E126Q116T116T189Q19D19D47Q116V116V190Y23D23D48K117D117D69R65D65D127I119A119A191R65G65G128I119D119D192R65P65P129I119E119E193I66D66D130I119M119M194I66G66G131I119N119N195I66P66P132I119Q119Q196N68Q68Q51I119S119S197V69D69D133I119T119T198V69G69G134H120D120D71V69P69P135L123D123D72

[0051] The IL-21 mutein as described herein may comprise an amino acid sequence of any one of SEQ ID NOs: 47, 48, 51, 61, 62, 64-67, 69, 71-112, 114-198, 249-254, or 283. The IL-21 mutein may comprise an amino acid sequence which is at least at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to one of SEQ ID NOs: 47, 48, 51, 61, 62, 64-67, 69, 71-112, 114-198, 249-254, or 283.

[0052] Also described are IL-21 muteins comprising only two amino acid substitutions, relative to SEQ ID NO: 1, and the two amino acid substitutions occur at two of positions 5, 9, 15, 70, 71, 72, 73, and 76 of SEQ ID NO: 1. The IL-21 mutein may comprise only two amino acid substitutions, relative to SEQ ID NO: 1, and the two substitutions may occur at a pair of amino acid positions selected from the group consisting of: 5 and 76; 9 and 76; 15 and 70; 15 and 71; 15 and 72; 15 and 73; 70 and 73; 70 and 76; 71 and 73; 71 and 76; 72 and 73; 72 and 76; and 73 and 76. The IL-21 mutein may comprise any one of the amino acid sequences of SEQ ID NOs: 199-208 and 210-212. Also described are IL-21 muteins comprising only two amino acid substitutions, relative to SEQ ID NO: 1, and the two amino acid substitutions occur at two of positions 5, 9, 73, and 76 or SEQ ID NO: 1. One of the substitutions may occur at position 76 of SEQ ID NO: 1. The substitute amino acid at position 76 of SEQ ID NO: 1 may be an aliphatic amino acid or an acidic amino acid. The aliphatic amino acid may be alanine. The acidic amino acid may be aspartic acid or glutamic acid. The acidic amino acid may be glutamic acid. The IL-21 mutein may comprise a substitute amino acid at position 76 and an aliphatic amino acid or acidic amino acid at position 5, 9, or 73 of SEQ ID NO: 1. The substitute amino acid at position 5, 9, or 73 may be an aliphatic amino acid, an acidic amino acid, or an amino acid with a side chain amide. The aliphatic amino acid may be alanine, the acidic amino acid may be glutamic acid, and the amino acid with a side chain amide may be glutamine. The IL-21 mutein may comprise a substitute amino acid at position 76 of SEQ ID NO: 1 (optionally, an aliphatic amino acid or an acidic amino acid) and a substitute amino acid at position 5 or 9 (according to the numbering of SEQ ID NO: 1).

[0053] The IL-21 mutein as described herein may comprise an amino acid sequence of any one of the SEQ ID NOs: shown in Table C. TABLE CAmino Acid Substitutions1 st< Amino Acid position of SEQ ID NO: 11 st< Substitute Amino Acid2 nd< Amino Acid position of SEQ ID NO: 12 nd< Substitute Amino AcidSEQ ID NO:R5E, R76E5E76E239R5E, R76A5E76A238R5A, R76A5A76A236R5Q, R76A5Q76A240R5A, R76E5A76E237R5Q, R76E5Q76E241R9E, R76E9E76E245R9A, R76E9A76E243R9E, R76A9E76A244R9A, R76A9A76A242D15N, S70T15N70T213D15N, I71L15N71L214D15N, K72A15N72A215D15N, K73A15N73A216S70T, K73Q70T73Q219S70T, R76A70T76A246S70T, R76D70T76D247S70T, R76E70T76E248I71L, K73Q71L73Q217I71L, R76A71L76A227I71L, R76D71L76D228I71L, R76E71L76E229K72A, K73Q72A73Q218K72A, R76A72A76A230K72A, R76D72A76D231K72A, R76E72A76E232K73A, R76A73A76A233K73A, R76D73A76D234K73A, R76E73A76E235

[0054] The IL-21 mutein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 213-219 and 227-248. The IL-21 mutein may comprise an amino acid sequence which is at least at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to one of SEQ ID NOs: 213-219 and 227-248.Peptide Length

[0055] The IL-21 muteins described herein may comprise a peptide backbone of any number of amino acids, i.e., can be of any peptide length. The peptides described herein may be the about same length as SEQ ID NO: 1, i.e., are 133 (± about 1 to about 20, ± about 1 to about 15, ± about 1 to about 10, or ± about 1 to about 5) amino acids in length. The presently disclosed peptide may be longer than 133 amino acids in length by virtue of being fused to another polypeptide chain, e.g., an antibody heavy chain comprising about 400 to about 600 amino acids, an antibody light chain comprising about 150 to about 300 amino acids, as further described herein.Additional peptide modifications

[0056] Alternatively or additionally, the IL-21 mutein may be lipidated (e.g., myritoylated, palmitoylated), glycosylated, amidated, carboxylated, phosphorylated, esterified, acylated, acetylated, cyclized, or converted into an acid addition salt and / or optionally dimerized or polymerized, or conjugated, as further described herein.Pharmaceutically acceptable salts

[0057] The IL-21 mutein may be in the form of a salt, e.g., a pharmaceutically acceptable salt. Such salts can be prepared in situ during the final isolation and purification of the IL-21 mutein or separately prepared by reacting a free base function with a suitable acid. Examples of acids which can be employed to form pharmaceutically acceptable acid addition salts include, for example, an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, sulphuric acid, and phosphoric acid, and an organic acid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

[0058] Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, maleate, methane sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate.

[0059] Basic addition salts also can be prepared in situ during the final isolation and purification of the IL-21 mutein, or by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like, and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium, amongst others. Other representative organic amines useful for the formation of base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0060] Further, basic nitrogen-containing groups can be quaternized with such active agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.Purification

[0061] The IL-21 muteins as described herein can be purified. The term "purified" as used herein means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. The purity of the compound (e.g., in the composition) may be at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 98% or may be about 100%.Peptidomimetics

[0062] The IL-21 mutein may be a peptidomimetic, or at least a portion of the mutein may be peptidomimetic. Peptidomimetics as well as methods of making the same are known in the art. See, for example, Advances in Amino Acid Mimetics and Peptidomimetics, Volumes 1 and 2, ed., Abell, A., JAI Press Inc., Greenwich, CT, 2006. The peptidomimetic may be a D-peptide peptidomimetic comprising D-isomer amino acids. The peptidomimetic may be a peptoid in which the side chain of an amino acid is connected to the alpha nitrogen atom of the peptide backbone. Methods of making peptoids are known in the art. See, e.g., Zuckermann et al., JACS 114(26): 10646-10647 (1992) and Design, Synthesis, and Evaluation of Novel Peptoids, Fowler, Sarah, University of Wisconsin-Madison, 2008. The peptidomimetic may be a β-peptide comprising β amino acids which have their amino group bonded to the β-cargon rather than the alpha carbon. Methods of making β-peptides are known in the art. See, for example, Seebach et al., Helvetica Chimica Acta 79(4): 913-941 (1996).Binding Characteristics

[0063] The IL-21 muteins may bind to the IL-21 receptor (IL-21R) with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21R. The IL-21 muteins may bind to the human IL-21R with a reduced affinity, relative to the affinity of wild-type human IL-21 for the human IL-21R. The IL-21 muteins may bind to the alpha chain of the human IL-21R with a reduced affinity, relative to the affinity of wild-type human IL-21 for the alpha chain of the human IL-21R. IL-21 muteins which bind to the alpha chain of the human IL-21R with a reduced affinity, relative to the affinity of wild-type human IL-21 for the alpha chain of the human IL-21R may contain one, two, or more substitutions located at an amino acid position selected from the group consisting of: 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, and 80, according to the amino acid position numbering of SEQ ID NO: 1. Specific amino acid substitutions that can be made at such positions are discussed herein (see, e.g., Tables A, B, and C).

[0064] The IL-21 muteins may bind to the gamma chain of human IL-21R with a reduced affinity, relative to the affinity of wild-type human IL-21 for the gamma chain of the human IL-21R. IL-21 muteins which bind to the gamma chain of human IL-21R with a reduced affinity, relative to the affinity of wild-type human IL-21 for the gamma chain of the human IL-21R may contain one, two, or more substitutions located at an amino acid position selected from the group consisting of: 11, 14, 15, 109, 110, 112, 113, 116, 117, 119, 120 and 123, according to the amino acid position numbering of SEQ ID NO: 1. Specific amino acid substitutions that can be made at such positions are discussed herein (see, e.g., Tables A, B, and C).

[0065] The IL-21 muteins may bind to the gamma chain of human IL-21R with a reduced affinity, relative to the affinity of wild-type human IL-21 for the alpha chain of the human IL-21R. The IL-21 muteins may bind to the cynomolgus monkey IL-21R with a reduced affinity, relative to the affinity of wild-type cynomolgus IL-21 for the cynomolgus IL-21R. The IL-21 muteins may bind to the alpha chain of the cynomolgus monkey IL-21R with a reduced affinity, relative to the affinity of wild-type cynomolgus IL-21 for the alpha chain of the cynomolgus IL-21R. The IL-21 muteins may bind to the gamma chain of cynomolgus monkey IL-21R with a reduced affinity, relative to the affinity of wild-type cynomolgus IL-21 for the gamma chain of the cynomolgus IL-21R. The IL-21 muteins may bind to the gamma chain of cynomolgus monkey IL-21R with a reduced affinity, relative to the affinity of wild-type cynomolgus IL-21 for the alpha chain of the cynomolgus IL-21R.

[0066] The IL-21 muteins described herein bind to IL-21R in a non-covalent and reversible manner. The binding strength of the muteins to IL-21R may be described in terms of its affinity, a measure of the strength of interaction between the binding site of the mutein and the IL-21R. The IL-21 muteins described herein may have high-affinity for IL-21R and thus will bind a greater amount of IL-21R in a shorter period of time than low-affinity IL-21 muteins. The IL-21 muteins described herein may have low-affinity for IL-21R and thus will bind a lesser amount of IL-21R in a longer period of time than high-affinity IL-21 muteins. The IL-21 mutein may have an equilibrium association constant, KA, which is at least 10 5< M -1< , at least 10 6< M -1< , at least 10 7< M -1< , at least 10 8< M -1< , at least 10 9< M -1< , or at least 10 10< M -1< . As understood by the artisan of ordinary skill, KA can be influenced by factors including pH, temperature and buffer composition.

[0067] The binding strength of the IL-21 mutein to IL-21R may be described in terms of its sensitivity. K D is the equilibrium dissociation constant, a ratio of k off / k on , between the IL-21 mutein and IL-21R. K D and KA are inversely related. The K D value relates to the concentration of the mutein (the amount of mutein needed for a particular experiment) and so the lower the K D value (lower concentration needed) the higher the affinity of the mutein. The binding strength of the IL-21 mutein to IL-21R may be described in terms of K D . The K D of the IL-21 muteins described herein may be about 10 -1< M, about 10 -2< M, about 10 -3< M, about 10 -4< M, about 10 -5< M, about 10 -6< M, or less. The K D of the IL-21 muteins described herein may be micromolar, nanomolar, picomolar or femtomolar. The K D of the IL-21 muteins described herein may be within a range of about 10 -4< to 10 -6< M, or 10 -7< to 10 -9< M, or 10 -10< to 10 -12< M, or 10 -13< to 10 -15< M. The IL-21 mutein may bind to the human IL-21R with a K D that is greater than or is about 0.04 nM. The IL-21 mutein may bind to the human IL-21R with a K D of about 0.01 nM to about 20 nM, 0.02 nMto 20 nM, 0.05 nMto 20 nM, 0.05 nMto 15 nM, 0.1 nMto 15 nM, 0.1 nMto 10 nM, 1 nM to 10 nM, or 5 nM to 10 nM. The IL-21 mutein may bind to the cynomolgus monkey IL-21R with a K D that is greater than or is about 0.055 nM. The IL-21 mutein may bind to the cynomolgus monkey IL-21R with a K D of about 0.01 nM to about 20 nM, 0.02 nM to 20 nM, 0.05 nM to 20 nM, 0.05 nM to 15 nM, 0.1 nM to 15 nM, 0.1 nM to 10 nM, 1 nM to 10 nM, or 5 nM to 10 nM.

[0068] The IL-21 mutein may exhibit a reduction in binding affinity for IL-21R α-chain. The IL-21 mutein may be a mutein (e.g., single or double) that exhibits about a 2-, 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100-, 105-, 110-, 115-, 120-, 125-, 130-, 135-, 140-, 145-, 150-, 175-, 200-, 225-, 250-, 275-, 300-, 325-, 350-, 375-, 400-, 425-, 450-, 475-, 500-, 525-, 550-, 575-, 600-, 625-, 650-, 675-, 700-, 725-, 750-, 775-, 800-, 825-, 850-, 875-, 900-, 925-, 950-, 975-fold, 1000-fold, or more reduction in binding affinity for IL-21R α-chain. The IL-21 mutein may be a double mutein exhibiting the reduction in binding affinity for IL-21R α-chain.

[0069] The above reduction in binding affinity of the IL-21 mutein (e.g., single or double mutein) may result in a reduced affinity for IL-21R α-chain compared to the affinity of about 0.025 nM of wild-type human IL-21 for IL-21R α-chain. Accordingly, a 2-fold reduction in affinity as discussed above would result in an IL-21 mutein with an affinity of about 0.05 nM for IL-21R α-chain. Thus, the IL-21 mutein (e.g., single or double) may have an affinity of about 0.05, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1.0, 1.125, 1.25, 1.375, 1.5, 1.625, 1.75, 1.875, 2.0, 2.125, 2.25, 2.375, 2.5, 2.625, 2.75, 2.875, 3.0, 3.125, 3.25, 3.375, 3.5, 3.625, 3.75, 4.375, 5, 5.625, 6.25, 6.875, 7.5, 8.125, 8.75, 9.375, 10.0, 10.625, 11.25, 11.875, 12.5, 13.125, 13.75, 14.375, 15.0, 15.625, 16.25, 16.875, 17.5, 18.125, 18.75, 19.375, 20.0, 20.625, 21.25, 21.875, 22.5, 23.125, 23.75, 24.375, 25 nM, or more for IL-21R α-chain. The IL-21 mutein may be a double mutein exhibiting a reduced binding affinity for IL-21R α-chain.

[0070] The IL-21 mutein may exhibit a reduction in activity as measured by an in vitro STAT3 phosphorylation assay. The IL-21 mutein may be a mutein (e.g., single or double) that exhibits about a 2-, 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100-, 105-, 110-, 115-, 120-, 125-, 130-, 135-, 140-, 145-, 150-, 175-, 200-, 225-, 250-, 275-, 300-, 325-, 350-, 375-, 400-, 425-, 450-, 475-, 500-, 525-, 550-, 575-, 600-, 625-, 650-, 675-, 700-, 725-, 750-, 775-, 800-, 825-, 850-, 875-, 900-, 925-, 950-, 975-fold, 1000-fold, or more reduction in activity as measured by a STAT3 phosphorylation assay. The IL-21 mutein may be a double mutein exhibiting the reduction in in activity as measured by a STAT3 phosphorylation assay.IL-21 mutein conjugates

[0071] Also described are conjugates comprising one or more of the IL-21 muteins as described herein linked to a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with the term "conjugate moiety" and refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the IL-21 muteins described herein. Exemplary conjugate moieties that can be linked to any of the IL-21 muteins described herein include but are not limited to a heterologous peptide or polypeptide (including for example, an immunoglobulin or portion thereof (e.g., variable region, CDR, or Fc region)), a targeting agent, a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents. A conjugate is described comprising an IL-21 mutein as described herein and an immunoglobulin. The conjugate may comprise one or more of the IL-21 muteins described herein and one or more of: a peptide (which is distinct from the IL-21 muteins described herein), a polypeptide, a nucleic acid molecule, an antibody or fragment thereof, a polymer, a quantum dot, a small molecule, a toxin, a diagnostic agent, a carbohydrate, an amino acid.

[0072] The conjugate as described herein may comprise an IL-21 mutein as described herein and a heterologous moiety which is a polypeptide (e.g., a polypeptide distinct from any of the IL-21 muteins described herein), and the conjugate may be a fusion polypeptide or fusion protein or a chimeric protein or chimeric polypeptide. Additional descriptions of such conjugates are provided herein under "Fusion proteins ".

[0073] The heterologous moiety may be attached via non-covalent or covalent bonding to the IL-21 mutein as described herein. The linkage between the IL-21 mutein and the heterologous moiety may be achieved via covalent chemical bonds, e.g., peptide bonds, disulfide bonds, and the like, or via physical forces, such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.

[0074] A variety of non-covalent coupling systems may be used, including, e.g., biotin-avidin, ligand / receptor, enzyme / substrate, nucleic acid / nucleic acid binding protein, lipid / lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.

[0075] The IL-21 mutein may be linked to a conjugate moiety via direct covalent linkage by reacting targeted amino acid residues of the IL-21 mutein with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of these targeted amino acids. Reactive groups on the IL-21 mutein or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido or hydrazino group. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art. Alternatively, the conjugate moieties can be linked to the IL-21 mutein indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier.

[0076] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid, chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0077] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0078] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

[0079] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[0080] The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

[0081] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0082] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine, and / or amidation or esterification of the C-terminal carboxylic acid group.

[0083] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the IL-21 mutein. Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO87 / 05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0084] The heterologous moiety may be attached to the IL-21 mutein as described herein via a linker. The linker may comprise a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. The chain atoms may be all carbon atoms. The chain atoms in the backbone of the linker may be selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate. The linker may provide a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. The length of the linker may be long enough to reduce the potential for steric hindrance. If the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptidyl linkers may be any length. Exemplary peptidyl linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length, and are flexible or rigid. The linker may be a peptide comprising about 2 to about 20 amino acids. The linker may be a peptide comprising about 2 to about 15 amino acid, about 2 to about 10 amino acids, or about 2 to about 5 amino acids. Suitable peptide linkers are known in the art. See, e.g., Chen et al., Adv Drug Delivery Reviews 65(10): 1357-1369 (2013); Arai et al., Protein Eng Des Sel 14(8): 529-532 (2001); and Wriggers et al., Curr Trends in Peptide Science 80(6): 736-746 (2005). The linker may be a peptide comprising the amino acid sequence GGGGS (SEQ ID NO: 262).Fusion proteins

[0085] The IL-21 mutein may be linked to a polypeptide which is distinct from any of the IL-21 muteins described herein, and the conjugate may be a fusion polypeptide or fusion protein or a chimeric protein or chimeric polypeptide. Accordingly, described herein are fusion polypeptides or fusion proteins comprising an IL-21 mutein as described herein and a heterologous polypeptide or peptide. The fusion protein as described herein may comprise an IL-21 mutein as described herein linked to an antigen-binding protein. The antigen-binding protein may be an antibody or immunoglobulin, or an antigen binding antibody fragment thereof, or an antibody protein product.

[0086] Collectively, antibodies form a family of plasma proteins known as immunoglobulins and comprise of immunoglobulin domains. (Janeway et al., Immunobiology: The Immune System in Health and Disease, 4th ed., Elsevier Science Ltd. / Garland Publishing, 1999. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions. For example, an antibody may be an IgG which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0087] The general structure and properties of CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0088] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. The present disclosure includes all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, the antibody may be an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0089] The antibody can be a monoclonal antibody or a polyclonal antibody. The antibody may comprise a sequence that is substantially similar to a naturally-occurring antibody produced by a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, and the like. In this regard, the antibody can be considered as a mammalian antibody, e.g., a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, and the like. The antibody may be a human antibody. The antibody may be a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody containing domains from two or more different antibodies. A chimeric antibody can, for example, contain the constant domains from one species and the variable domains from a second, or more generally, can contain stretches of amino acid sequence from at least two species. A chimeric antibody also can contain domains of two or more different antibodies within the same species. The term "humanized" when used in relation to antibodies refers to antibodies having at least CDR regions from a non-human source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies. For example, humanizing can involve grafting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence more similar to a human sequence.

[0090] An antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab') 2 fragment and a pFc' fragment. The fusion protein of the present disclosure may comprise an antigen binding antibody fragment. As used herein, the term "antigen binding antibody fragment refers to a portion of an antibody molecule that is capable of binding to the antigen of the antibody and is also known as "antigen-binding fragment" or "antigen-binding portion". The antigen binding antibody fragment may be a Fab fragment or a F(ab') 2 fragment.

[0091] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as "antibody protein products". Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs and VHH / VH (discussed below). The smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble, flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are VHH / VH of camelid heavy chain Abs as well as single domain Abs (sdAb). The building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ∼15 amino acid residues. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well-described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012).

[0092] Other antibody protein products include a single chain antibody (SCA); a diabody; a triabody; a tetrabody; bispecific or trispecific antibodies, and the like. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015).

[0093] The fusion protein as described herein may comprise any one of these antibody protein products. The fusion protein as described herein may comprise any one of an scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy chain antibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0094] The fusion protein as described herein may comprise an antibody protein product in monomeric form, or polymeric, oligomeric, or multimeric form. Where the antibody comprises two or more distinct antigen binding regions fragments, the antibody may be considered bispecific, trispecific, or multi-specific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the antibody.

[0095] The antibody, antigen binding antibody fragment or antibody protein product may bind to a tumor antigen. The tumor antigen may be an antigen derived from a viral protein, an antigen derived from point mutations, or an antigen encoded by a cancer-germline gene. The tumor antigen may be p53, KRAS, NRAS, MAGEA, MAGEB, MAGEC, BAGE, GAGE, LAGE / NY-ESO1, SSX, tyrosinase, gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1, TRP2, CEA, RAGE-1, HER2 / NEU, WT1. The antibody, antigen binding antibody fragment or antibody protein product of the fusion protein as described herein may bind to an immunotherapy agent or is an immunotherapy agent, as described herein. The antibody, antigen binding antibody fragment or antibody protein product of the fusion protein as described herein may bind to a cytokine, lymphokine, growth factor, or hematopoietic factor, as described herein.

[0096] The fusion protein as described herein may comprise a cytokine (e.g., an IL-21 mutein described herein) and an antibody, antigen binding antibody fragment thereof or antibody protein product, which binds to a protein of the immune checkpoint pathway, a tumor antigen, a cytokine, lymphokine, growth factor, or other hematopoietic factor, including but not limited to any of those described herein. The fusion protein of the present disclosure may comprise a cytokine (e.g., an IL-21 mutein described herein) and an antibody, antigen binding antibody fragment thereof or antibody protein product, which binds to a protein of the immune checkpoint pathway selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or co-stimulatory receptor: ICOS, OX40, 41BB, CD27, GITR.

[0097] The fusion protein of the present disclosure may comprise a cytokine and an antibody (or antigen binding antibody fragment thereof) which binds to a protein of the immune checkpoint pathway. Suitable cytokines include, for example, cytokines that enhance TH-1-type responses; and cytokines that activate STAT 1, 3, 4, or 5. The cytokine may be an interleukin. The cytokine may be an interleukin that enhances T cell activity such as, for example, IL-2, IL-7, IL-10, IL-12, IL-15, or IL-21. Such cytokines can be modified (e.g., via mutations) to attenuate their affinity for their respective receptor. Such muteins can exhibit improved safety profiles by reducing off-target and unwanted interactions. Thus, the cytokines can be modified to generate IL-2, IL-7, IL-10, IL-12, IL-15, or IL-21 muteins. The cytokine may be an IL-21 mutein described herein. Suitable antibodies (or antigen binding antibody fragments thereof) which binds to a protein of the immune checkpoint pathway include, for example, those which bind CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, LAG3, CD112 TIM3, BTLA, or co-stimulatory receptor: ICOS, OX40, 41BB, or GITR. The antibody (or antigen binding antibody fragment thereof) may bind to PD-1 (e.g., human PD-1).

[0098] The fusion protein as described herein may be a multispecific fusion protein which comprises a cytokine, an antibody (or antigen binding antibody fragment thereof), and at least one additional targeting moiety. For example, the fusion protein as described herein may be a trispecific fusion protein which comprises a cytokine, an antibody (or antigen binding antibody fragment thereof), and one additional targeting moiety.

[0099] The fusion protein as described herein may comprise an IL-21 mutein described herein and a PD-1 binding antagonist. The term "PD-1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. The PD-1 binding antagonist may be a molecule that inhibits the binding of PD-1 to one or more of its binding partners. The PD-1 binding antagonist may inhibit the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding antibody fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. A PD-1 binding antagonist may reduce the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). The PD-1 binding antagonist may be an anti-PD-1 antibody. Examples of anti-PD-1 antibodies include nivolumab (BMS-936558), pembrolizumab (MK-3475), BMS 936558, BMS- 936559, TSR-042 (Tesaro), ePDR001 (Novartis), and pidilizumab (CT-011). Additional specific examples of PD-1 binding antagonists are provided infra.

[0100] The PD-1 binding antagonist may comprise, consist essentially of, or consist of an antigen-binding protein which binds to PD-1. The antigen-binding protein may be an antibody, antigen binding antibody fragment thereof, or an antibody protein product, which binds to PD-1.

[0101] The fusion protein as described herein may comprise an IL-21 mutein, as described herein, and an anti-PD-1 antibody (as described herein), an antigen binding antibody fragment thereof, or an anti-PD-1 antibody protein product. The anti-PD-1 antibody may be a monoclonal IgG. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may be a monovalent or bivalent. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may bind to human PD-1, which has the amino acid sequence of SEQ ID NO: 263. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may bind to cynomolgus PD-1, which has the amino acid sequence of SEQ ID NO: 264. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may bind to both human PD-1 and cynomolgus PD-1. The fusion protein comprises an IL-21 mutein (as described herein) and an anti-PD-1 antibody (as described herein).

[0102] The binding strength of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product to PD-1 may be described in terms of K D . The K D of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product provided herein may be about 10 -1< M, about 10 -2< M, about 10 -3< M, about 10 -4< M, about 10 -5< M, about 10 -6< M, about 10 -7< M, about 10 -8< M, about 10 -9< M, or less. The K D of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product as described herein may be micromolar, nanomolar, picomolar or femtomolar. The K D of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product as described herein may be within a range of about 10 -4< to 10 -6< M, or 10 -7< to 10 -9< M, or 10 -10< to 10 -12< M, or 10 -13< to 10 -15< M. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may have high affinity for human PD-1, cynomolgus PD-1, or both. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may have a K D for human PD-1 of less than 100 pM, optionally, about 1 pM to about 50 pM. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may have a K D for human PD-1 within about 1 pM to about 20 pM or less than about 10 pM. The anti-PD-1 antibody, a antigen binding ntibody fragment thereof, or anti-PD-1 antibody protein product may have a K D for cynomolgus PD-1 of less than 100 pM, optionally, about 1 pM to about 75 pM. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may have a K D for cynomolgus PD-1 within about 1 pM to about 20 pM or less than 10 pM.

[0103] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may be a PD-1 binding antagonist that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may block PD-1 from binding to its ligand PD-L1 or PD-L2. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may inhibit at least 50% of the binding interactions between PD-1 and PD-L1 or PD-L2. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may exhibit at least about 50%, at least about 60%, or at least about 70% inhibition of the binding interaction between PD-1 and PD-L1 or PD-L2.

[0104] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may inhibit PD-1-mediated production of IL-2 by T cells in a mixed lymphocyte reaction (MLR). The IC 50 of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product in the MLR may be within about 0.1 nM to about 5 nM. The IC 50 of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product in the MLR may be less than 2 nM or less than 1 nM. The IC 50 of the anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product in the MLR may be about 0.5 nM to about 2 nM.

[0105] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence selected from the group consisting of: SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372, and 382, (see Table D) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) an HC CDR2 amino acid sequence selected from the group consisting of: SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373, and 383, (see Table D) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) an HC CDR3 amino acid sequence selected from the group consisting of: SEQ ID NOs: 314, 324, 334, 344, 3545, 364, 374, and 384, (see Table D) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence selected from the group consisting of: 315, 325, 335, 345, 355, 365, 375, and 385, (see Table D) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (e) an LC CDR2 amino acid sequence selected from the group consisting of: 316, 326, 336, 346, 356, 366, 376, and 386, (see Table D) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (f) an LC CDR3 amino acid sequence selected from the group consisting of: 317, 327, 337, 347, 357, 367, 377, and 387, (see Table D) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (g) a combination of any two, three, four, five, or six of (a)-(f). The anti-PD-1 antibody protein product may comprise such CDRs. TABLE D20A2 20C1 22D4 20C1.006 20C1.009 20A2.003 22D4.006 22D4.017 HC CDR1312322332342352362372382HC CDR2313323333343353363373383HC CDR3314324334344354364374384LC CDR1315325335345355365375385LC CDR2316326336346356366376386LC CDR3317327337347357367377387Number represents the relevant SEQ ID NO.

[0106] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table D and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table D. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, and a HC CDR3 amino acid sequence set forth in Table D and at least 1 or 2 of the LC CDR amino acid sequences set forth in Table D. The anti-PD-1 antibody protein product may comprise such CDRs.

[0107] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise 3, 4, 5, or all 6 of the amino acid sequences designated by the SEQ ID NOs: in a single column of Table D. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise each of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single column of Table D and at least 1 or 2 of the HC CDR amino acid sequences designated by the SEQ ID NOs: in the same single column or another single column of Table D. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise each of the HC CDR amino acid sequences designated by the SEQ ID NOs: of a single column of Table D and at least 1 or 2 of the LC CDR amino acid sequences designated by the SEQ ID NOs: in the same single column or another single column of Table D. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 312-317; (b) SEQ ID NOs: 322-327; (c) SEQ ID NOs: 332-337; (d) SEQ ID NOs: 342-347; (e) SEQ ID NOs: 352-357; (f) SEQ ID NOs: 362-367; (g) SEQ ID NOs: 372-377; and (h) SEQ ID NOs: 382-387. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise all 6 of the CDR amino acid sequences in Table D for any one of the 20A2, 20C1, 22D4, 20C1.006, 20C1.009, 20A2.003, 22D4.006, or 22D4.017 antibodies. The anti-PD-1 antibody protein product may comprise such CDRs.

[0108] The amino acid sequences of Table D may be separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). There may be about 10 to about 20 amino acids between the sequences of the LC CDR1 and the LC CDR2 and about 25 to about 40 amino acids between the sequences of the LC CDR2 and the LC CDR3. There may be about 14 to about 16 amino acids between the sequences of the LC CDR1 and the LC CDR2 and about 30 to about 35 amino acids between the sequences of LC CDR2 and the LC CDR3. There may be about 10 to about 20 amino acids between the sequences of the HC CDR1 and HC CDR2 and about 25 to about 40 amino acids between the sequences of the HC CDR2 and the HC CDR3. There may be about 14 to about 16 amino acids between the sequences of the HC CDR1 and HC CDR2 and about 30 to about 35 amino acids between the sequences of the HC CDR2 and HC CDR3.

[0109] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise (a) a heavy chain variable region amino acid sequence selected from the group consisting of: 318, 328, 338, 348, 358, 368, 378, and 388, (see Table E) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (b) a light chain variable region amino acid sequence selected from the group consisting of: 319, 329, 339, 349, 359, 369, 379, and 389, (see Table E) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (c) both (a) and (b). The anti-PD-1 antibody protein product may comprise such variable regions. TABLE E20A2 20C1 22D4 20C1.006 20C1.009 20A2.003 22D4.006 22D4.017 HC VARIABLE318328338348358368378388LC VARIABLE319329339349359369379389HC (full length)320330340350360370380390LC (full length)321331341351361371381391Number represents the relevant SEQ ID NO.

[0110] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389.

[0111] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise (a) a heavy chain amino acid sequence selected from the group consisting of: 320, 330, 340, 350, 360, 370, 380, and 390, (see Table E) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (b) a light chain amino acid sequence selected from the group consisting of: 321, 331, 341, 351, 361, 371, 381, and 391, (see Table E) or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (c) both (a) and (b). The anti-PD-1 antibody protein product may comprise such variable regions.

[0112] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391. The anti-PD-1 antibody protein product may comprise such regions.

[0113] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) heavy chain amino acid sequences may comprise a set of charge pair mutations, as described herein. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) heavy chain amino acid sequences may comprise charge pair mutations selected from V1, V103, and V131 charge pair mutations.

[0114] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise an amino acid sequence which is similar to an above-referenced amino acid sequence, yet the antigen-binding protein substantially retains its biological function, e.g., its ability to bind to PD-1, e.g., human PD-1, cynomolgus PD-1, or to decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 or PD-L2.

[0115] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise an amino acid sequence which differs by only 1, 2, 3, 4, 5, 6, or more amino acids, relative to the above-referenced amino acid sequence(s). The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a variant sequence of the referenced sequence, which variant sequence differs by only one or two amino acids, relative to the referenced sequence. The antigen-binding protein may comprise one or more amino acid substitutions that occur outside of the CDRs, e.g., the one or more amino acid substitutions occur within the framework region(s) of the heavy or light chain. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise one or more amino acid substitutions yet the antigen-binding protein retains the amino acid sequences of the six CDRs. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise an amino acid sequence having only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions, relative to the above-referenced amino acid sequence(s).

[0116] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise an amino acid sequence which has greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the above-referenced amino acid sequence(s). The antigen-binding protein may comprise an amino acid sequence which has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to the above-referenced amino acid sequence. The antigen-binding protein may comprise an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity along the full-length of the above-referenced amino acid sequence. The antigen-binding protein may comprise an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the full-length of the above-referenced amino acid sequence.

[0117] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a variant sequence of the referenced sequence, which variant sequence has at least or about 70% sequence identity, relative to the above-referenced sequence. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a variant sequence of the referenced sequence, which variant sequence has at least or about 80% sequence identity, relative to the above-referenced sequence. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a variant sequence of the referenced sequence, which variant sequence has at least or about 90% sequence identity, relative to the above-referenced sequence. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a variant sequence of the referenced sequence, which variant sequence has at least or about 95% sequence identity, relative to the above-referenced sequence.

[0118] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise one, two, three, four, or five sequences of the SEQ ID NOs. in a single column of Table D and at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 312-387. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise one, two, three, four, or five sequences of a set of sequences selected from: (a) SEQ ID NOs: 312-317; (b) SEQ ID NOs: 322-327; (c) SEQ ID NOs: 332-337; (d) SEQ ID NOs: 342-347; (e) SEQ ID NOs: 352-357; (f) SEQ ID NOs: 362-367; (g) SEQ ID NOs: 372-377; and (h) SEQ ID NOs: 382-387, wherein the antibody or fragment thereof may further comprise at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to at least one of the sequences of the set. For instance, the anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise four sequences of SEQ ID NOs: 312-317, namely, SEQ ID NOs: 312-315, wherein the antibody or fragment thereof may comprise two variant sequences: one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%) sequence identity to SEQ ID NO: 316 and another variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO: 317. The anti-PD-1 antibody protein product may comprise such regions.

[0119] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) product may comprise a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 318, 319, 328, 329, 338, 339, 348, 349, 358, 359, 368, 369, 378, 379, 388, and 389. The antibody or fragment thereof may comprise a pair of variant sequences which have at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of sequences: one sequence of Table E and another sequence which is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 318, 319, 328, 329, 338, 339, 348, 349, 358, 359, 368, 369, 378, 379, 388, and 389. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of sequences: one sequence selected from (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389, and another sequence which may be a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to a sequence of (a) - (u). For instance, the anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise the sequence of SEQ ID NO: 318 and may further comprise a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO 319. The anti-PD-1 antibody protein product may comprise such regions.

[0120] The anti-PD-1 antibody (or antigen binding antibody fragment thereof may comprise a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 320, 321, 330, 331, 340, 341, 350, 351, 360, 361, 370, 371, 380, 381, 390, and 391. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of variant sequences which have at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of sequences: one sequence of Table E and another sequence which may be a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to any of SEQ ID NOs: 320, 321, 330, 331, 340, 341, 350, 351, 360, 361, 370, 371, 380, 381, 390, and 391. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of sequences: one sequence selected from (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391, and another sequence which may be a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to a sequence of (a) - (u). For instance, the anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise the sequence of SEQ ID NO: 320 and may further comprise a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity to SEQ ID NO 321. The anti-PD-1 antibody protein product may comprise such regions.

[0121] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise one or more amino acid modifications, relative to the naturally-occurring counterpart, in order to improve half-life / stability or to render the antibody more suitable for expression / manufacturability (e.g., as a fusion protein with the IL-21 mutein). The anti-PD-1 antibody may be designed to prevent or reduce interaction between the anti-PD-1 antibody and Fc receptors. The anti-PD-1 antibody may be a Stable Effector Functionless (SEFL) antibody comprising a constant region that lacks the ability to interact with Fcγ receptors. SEFL antibodies are known in the art. See, e.g., Liu et al., J Biol Chem 292: 1876-1883 (2016); and Jacobsen et al., J. Biol. Chem. 292: 1865-1875 (2017). The SEFL antibody may comprise one or more of the following mutations, numbered according to the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. The SEFL antibody may comprise N297G. The SEFL antibody may comprise A287C, N297G, and L306C. The SEFL antibody may comprise R292C, N297G, and V302C (i.e., SEFL2-2).

[0122] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise other half-life extension (HLE) modifications. The HLE modification may occur in the heavy chain constant region and may comprise one or more of the following mutations, numbered according to the EU system: M252Y, S254T, and T256E. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise one or two of M252Y, S254T, and T256E. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise all three of M252Y, S254T, and T256E. The heavy chain constant region may comprise an amino acid sequence of SEQ ID NO: 545 or SEQ ID NO: 547 or SEQ ID NO: 549 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 545 or SEQ ID NO: 547 or SEQ ID NO: 549. The HLE modification may occur in the heavy chain constant region and comprises one or more of the following mutations, numbered according to the EU system: L309D, Q311H, and N434S. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise one, two or all three of L309D, Q311H, and N434S. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise all three of L309D, Q311H, and N434S. The heavy chain constant region may comprise an amino acid sequence of SEQ ID NO: 544 or SEQ ID NO: 546 or SEQ ID NO: 548 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 544 or SEQ ID NO: 546 or SEQ ID NO: 548.

[0123] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise SEFL2-2 modifications and HLE modifications. The HLE modifications may comprise one or two or all three of M252Y, S254T, and T256E. The heavy chain constant region may comprise an amino acid sequence of SEQ ID NO: 551 or SEQ ID NO: 553 or SEQ ID NO: 555 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 551 or SEQ ID NO: 553 or SEQ ID NO: 555. The HLE modifications may comprise one or two or all three of L309D, Q311H, and N434S. The heavy chain constant region may comprise an amino acid sequence of SEQ ID NO: 550 or SEQ ID NO: 552 or SEQ ID NO: 554 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 550 or SEQ ID NO: 552 or SEQ ID NO: 554. The heavy chain additionally may comprise charge pair mutations as described below.

[0124] In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which glycans are attached to the asparagine of the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid except proline, and (2) O-linked glycosylation in which glycans are attached to serine or threonine. N-linked glycosylation begins in the Endoplasmic Reticulum (ER), where a complex set of reactions result in the attachment of a core glycan structure made essentially of two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified by action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible to the enzymes, it typically stays in the original HM form. If enzymes can access the saccharide, then many of the Man residues are cleaved off and the saccharide is further modified, resulting in the complex type N-glycans structure. For example, mannosidase-1 located in the cis-Golgi, can cleave or hydrolyze a HM glycan, while fucosyltransferase FUT-8, located in the medial-Golgi, fucosylates the glycan (Hanrue Imai- Nishiya (2007), BMC Biotechnology, 7:84). In exemplary aspects, the anti-PD-1 antibody is N-glycosylated, e.g., comprises one or more sugar moieties (e.g., glycans, saccharides) covalently attached to a specific amino acid of the heavy chain. In alternative aspects, the anti-PD-1 antibody is not glycosylated or does not comprise any sugar moieties (e.g., glycans, saccharides) covalently attached to a specific amino acid of the heavy chain.

[0125] The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284. The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, and further comprises a linker. The linker may comprise the amino acid sequence of SEQ ID NO: 262. Thus, the anti-PD-1 antibody may comprise a heavy chain constant region amino acid sequence of SEQ ID NO: 287, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 287. The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, with the C-terminal Lys clipped or removed. In this regard, the anti-PD-1 antibody may comprise a heavy chain constant region lacking the C-terminal Lys and may comprise the amino acid sequence of SEQ ID NO: 285, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 285. In general, the C-terminal lysine of an antibody undergoes cleavage by carboxypeptidase during expression. A heavy chain constant region lacking the C-terminal Lys advantageously prevents carboxypeptidase to act on the heavy chain of the anti-PD-1 antibody. The anti-PD-1 antibody may comprise a heavy chain constant region lacking the C-terminal Lys and further may comprise a linker. The linker may comprise the amino acid sequence of SEQ ID NO: 262. Thus, the anti-PD-1 antibody may comprise a heavy chain constant region amino acid sequence of SEQ ID NO: 286, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 286. The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, and with one or more SEFL mutations which prevent or reduce interaction between the anti-PD-1 antibody and Fc receptors, including but not limited to L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. The SEFL mutations may be SEFL2-2 mutations: R292C, N297G, and V302C, such that the anti-PD-1 antibody may comprise a heavy chain constant region amino acid sequence of SEQ ID NO: 265, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 265. The anti-PD-1 antibody may comprise a heavy chain constant region with SEFL2-2 mutations and with the C-terminal Lys clipped or removed. Such a heavy chain constant region may comprise the sequence of SEQ ID NO: 266. The anti-PD-1 antibody may comprise a heavy chain constant region with SEFL2-2 mutations, the C-terminal Lys clipped or removed, and a linker. Such a heavy chain constant region may comprise the sequence of SEQ ID NO: 267. The anti-PD-1 antibody may comprise a heavy chain constant region with SEFL2-2 mutations and a linker without the C-terminal Lys clipped. Such a heavy chain constant region may comprise the sequence of SEQ ID NO: 282.

[0126] The IL-21 mutein may be attached to the Fc of the anti-PD-1 antibody. The IL-21 mutein may be attached to one of the two heavy chains of the antibody. The IL-21 mutein may be attached to the C-terminus of one of the two heavy chains of the antibody.

[0127] The fusion protein may comprises only one IL-21 mutein (i.e., the fusion protein comprises an IL-21 mutein monomer). The IL-21 mutein may be attached to the C-terminus of one of the two heavy chains of the antibody. When the fusion protein comprises only one IL-21 mutein, the Fc of the antibody may comprise modifications designed to drive heterodimerization of the two heavy chains (one heavy chain fused to the IL-21 mutein and one heavy chain lacking the IL-21 mutein). Such modifications include Fc mutations such as knobs-into-holes, DuoBodies, Azymetric, charge pair, HA-TF, SEEDbody, and modifications with differential protein A affinity. See, e.g., Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. Knobs-into-holes mutations include T366W in the first heavy chain, and T366S, L368A, and / or Y407V in the second heavy chain. See, e.g., Ridgway et al., Protein Eng., 9 (1996), pp. 617-621; and Atwell et al., J. Mol. Biol., 270 (1997), pp. 26-35. DuoBody mutations include F405L in the first heavy chain and K409R in the second heavy chain. See, e.g., Labrijn et al., Proc. Natl. Acad. Sci. U.S.A., 110 (2013), pp. 5145-5150. Azymetric mutations include T350V, L351Y, F405A, and / or Y407V in the first heavy chain, and T350V, T366L, K392L, and / or T394W in the second heavy chain. See, e.g., Von Kreudenstein et al., mAbs, 5 (2013), pp. 646-654. HA-TF mutations include S364H and / or F405A in the first heavy chain, and Y349T and / or T394F in the second heavy chain. See, e.g., Moore et al., mAbs, 3 (2011), pp. 546-557. SEEDbody mutations include IgG / A chimera mutations in the first heavy chain and IgG / A chimera mutations in the second heavy chain. See, e.g., Davis et al., Protein Eng. Des. Sel., 23 (2010), pp. 195-202. Differential protein A affinity mutations include H435R in one heavy chain and no mutations in the other heavy chain. See, e.g., US Patent No. 8,586,713.

[0128] In a particular example, the mutations are charge pair mutations. The following are examples of such charge pair mutations, numbered according to the EU system. Charge pair mutations include K409D in the first heavy chain and D399K in the second heavy chain; K392D in the first heavy chain and E356K in the second heavy chain; or both K409D and K392D in the first heavy chain and both D399K and E356K in the second heavy chain (the latter denoted as "V1" herein). See, e.g., Gunasekaran et al., J Biol Chem 285: 19637-19646 (2010). In another particular example, the chair pair mutations include K439D, K392D, and K409D in the first heavy chain; and E356K and D399K in the second heavy chain (denoted as "VI03" herein). In yet another particular example, the charge pair mutations include K360E, K370E, K392E, and K409D in the first heavy chain; and E357K and D399K in the second heavy chain (denoted as "V131" herein). Charge pair mutations may also include K370D in the first heavy chain and E357K in the second heavy chain; or all three of K409D, K392D, and K370D in the first heavy chain and all three of D399K, E357K, and E356K in the second heavy chain (the latter denoted as "V4" herein). Additional charge pair mutations also include D221E, P228E, and / or L368E in the first heavy chain and D221R, P228R, and / or K409R in the second heavy chain. See, e.g., Strop et al., J. Mol. Biol., 420 (2012), pp. 204-219.

[0129] Where the fusion protein comprises only one IL-21 mutein (i.e., the fusion protein comprises an IL-21 mutein monomer) and the heavy chain contains the V1 charge pair mutations, the IL-21 mutein may be attached to the heavy chain containing the K409D and K392D mutations (e.g., the IL-21 mutein may be attached to a heavy chain comprising SEQ ID NO: 294, 296, or 298), or the heavy chain containing the D399K and E356K mutations (e.g., the IL-21 mutein may be attached to a heavy chain comprising SEQ ID NO: 295, 297, or 299. The IL-21 mutein may be attached to the heavy chain containing the D399K and E356K mutations.

[0130] Where the fusion protein comprises only one IL-21 mutein (i.e., the fusion protein comprises an IL-21 mutein monomer) and the heavy chain contains the V4 charge pair mutations, the IL-21 mutein may be attached to the heavy chain containing the K409D, K392D, and K370D mutations (e.g., the IL-21 mutein is attached to a heavy chain comprising SEQ ID NO: 288, 290, or 292), or the heavy chain containing the D399K, E357K, and E356K mutations (e.g., the IL-21 mutein is attached to a heavy chain comprising SEQ ID NO: 289, 291. or 293). The IL-21 mutein may be attached to the heavy chain containing the D399K, E357K, and E356K mutations.

[0131] Where the fusion protein comprises only one IL-21 mutein (i.e., the fusion protein comprises an IL-21 mutein monomer) and the heavy chain contains the V103 charge pair mutations, the IL-21 mutein may be attached to the heavy chain containing the K439D, K392D, and K409D mutations (e.g., the IL-21 mutein is attached to a heavy chain comprising SEQ ID NO: 472, 474, or 476), or the heavy chain containing the E356K and D399K mutations (e.g., the IL-21 mutein is attached to a heavy chain comprising SEQ ID NO: 473, 475, or 477). The IL-21 mutein may be attached to the heavy chain containing the E356K and D399K mutations.

[0132] Where the fusion protein comprises only one IL-21 mutein (i.e., the fusion protein comprises an IL-21 mutein monomer) and the heavy chain contains the V131 charge pair mutations, the IL-21 mutein may be attached to the heavy chain containing the K360E, K370E, K392E, and K409D mutations (e.g., the IL-21 mutein is attached to a heavy chain comprising SEQ ID NO: 478, 480, or 482), or the heavy chain containing the E357K and D399K mutations (e.g., the IL-21 mutein is attached to a heavy chain comprising SEQ ID NO: 479, 481, or 483). The IL-21 mutein may be attached to the heavy chain containing the E357K and D399K mutations.

[0133] Thus, the anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a set of charge pair mutations, as described herein. The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise charge pair mutations selected from V1, V103, and V131 charge pair mutations.

[0134] The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, with one or more charge pair mutations, e.g., the V1, V4, V103, or V131 charge pair mutations. The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, with V1 charge pair mutations, wherein a first heavy chain constant region comprises K409 and K392D mutations and a second heavy chain constant region comprises D399K and E356K mutations. Such a first heavy chain constant region may comprise the sequence of SEQ ID NO: 294 and such a second heavy chain constant region may comprise the sequence of SEQ ID NO: 295. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed such that the first and second heavy chain constant regions may comprise SEQ ID NO: 296 and 297, respectively. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed and a linker such that the first and second heavy chain constant regions may comprise SEQ ID NO: 298 and 299, respectively. The anti-PD-1 antibody may comprise a heavy chain constant region comprising V1 charge pair mutations and SEFL2-2 mutations. Such a first heavy chain constant region comprising the V1 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 306 and such a second heavy chain constant region comprising the V1 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 307. Additional variations of such first and second heavy chains, including, e.g., heavy chains with the C-terminal Lys clipped or removed (SEQ ID NOs: 308 and 309) and with the C-terminal Lys clipped or removed with a linker (SEQ ID NOs: 310 and 311) are contemplated.

[0135] The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, with V4 charge pair mutations, wherein a first heavy chain constant region comprises K409, K392D, and K370D mutations and a second heavy chain constant region comprises D399K, E356K, and E357K mutations. Such a first heavy chain constant region may comprise the sequence of SEQ ID NO: 288 and such a second heavy chain constant region may comprise the sequence of SEQ ID NO: 289. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed such that the first and second heavy chain constant regions may comprise SEQ ID NO: 290 and 291, respectively. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed and a linker such that the first and second heavy chain constant regions may comprise SEQ ID NO: 292 and 293, respectively. The anti-PD-1 antibody may comprise a heavy chain constant region comprising V4 charge pair mutations and SEFL2-2 mutations. Such a first heavy chain constant region comprising the V4 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 300 and such a second heavy chain constant region comprising the V4 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 301. Additional variations of such first and second heavy chains, including, e.g., heavy chains with the C-terminal Lys clipped (SEQ ID NOs: 302 and 303) and with the C-terminal Lys clipped or removed with a linker (SEQ ID NOs: 304 and 305) are contemplated.

[0136] The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, with V103 charge pair mutations, wherein a first heavy chain constant region comprises the sequence of SEQ ID NO: 484 and a second heavy chain constant region comprises the sequence of SEQ ID NO: 485. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed such that the first and second heavy chain constant regions may comprise SEQ ID NO: 486 and 487, respectively. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed and a linker such that the first and second heavy chain constant regions may comprise SEQ ID NO: 488 and 489, respectively. The anti-PD-1 antibody may comprise a heavy chain constant region comprising V103 charge pair mutations and SEFL2-2 mutations. Such a first heavy chain constant region comprising the V103 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 484 and such a second heavy chain constant region comprising the V103 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 485. Additional variations of such first and second heavy chains, including, e.g., heavy chains with the C-terminal Lys clipped (SEQ ID NOs: 486 and 487) and with the C-terminal Lys clipped or removed with a linker (SEQ ID NOs: 488 and 489) are contemplated.

[0137] The anti-PD-1 antibody may comprise a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 284, with V131 charge pair mutations, wherein a first heavy chain constant region comprises the sequence of SEQ ID NO: 478 and a second heavy chain constant region comprises the sequence of SEQ ID NO: 479. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed such that the first and second heavy chain constant regions may comprise SEQ ID NO: 480 and 481, respectively. Such first and second heavy chain constant regions may have the C-terminal Lys clipped or removed and a linker such that the first and second heavy chain constant regions may comprise SEQ ID NO: 482 and 483, respectively. The anti-PD-1 antibody may comprise a heavy chain constant region comprising V131 charge pair mutations and SEFL2-2 mutations. Such a first heavy chain constant region comprising the V131 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 490 and such a second heavy chain constant region comprising the V131 charge pair mutations and SEFL2-2 mutations may comprise a sequence of SEQ ID NO: 491. Additional variations of such first and second heavy chains, including, e.g., heavy chains with the C-terminal Lys clipped (SEQ ID NOs: 492 and 493) and with the C-terminal Lys clipped or removed with a linker (SEQ ID NOs: 494 and 495) are contemplated.

[0138] The fusion protein may comprise more than one IL-21 mutein (i.e., the fusion protein comprises an IL-21 mutein dimer or IL-21 mutein multimer). The fusion protein may comprise 2, 3, or 4 (or more) IL-21 muteins. When the fusion protein comprises more than one IL-21 mutein, each IL-21 mutein may comprise the same structure, e.g., the same amino acid sequence. The fusion protein may comprise an IL-21 homodimer or IL-21 homomultimer. Each IL-21 mutein of the fusion protein may comprise a different structure, e.g., a different amino acid sequence. The fusion protein may comprise an IL-21 heterodimer or IL-21 heteromultimer. The fusion protein may comprise two IL-21 muteins, wherein the first IL-21 mutein is linked to the C-terminus of the first antibody heavy chain, and the second IL-21 mutein is linked to the C-terminus of the second antibody heavy chain. Each IL-21 mutein may have the same amino acid sequence (e.g., is an IL-21 mutein homodimer). The first IL-21 mutein may have a different amino acid sequence relative to the second IL-21 mutein (e.g., is an IL-21 mutein heterodimer).

[0139] With regard to the fusion proteins comprising one or more IL-21 muteins, each IL-21 mutein may be attached to one of the heavy chains of the antibody with or without a linker. The IL-21 mutein may be attached to the C-terminus of one of the antibody heavy chains via a linker and the linker is a peptide. The peptide may comprise the amino acid sequence of GGGGS (SEQ ID NO: 262). The IL-21 mutein may be directly attached to the C-terminus of one of the heavy chains of the antibody without a linker.

[0140] The fusion protein may comprise only one IL-21 mutein which is directly attached to the C-terminus of one of the heavy chains of the anti-PD-1 antibody. The IL-21 mutein may comprise an amino acid substitution listed in Table 4 or a sequence of a SEQ ID NO: listed in Table 4. The IL-21 mutein may comprise an amino acid substitution listed in Table 5 or a sequence of a SEQ ID NO: listed in Table 5. The IL-21 mutein may comprise the amino acid substitutions listed in Table 7 or a sequence of a SEQ ID NO: listed in Table 7. The IL-21 mutein may comprise the amino acid substitutions listed in any one of Tables 6 and 8-14 or a sequence of a SEQ ID NO: listed in these Tables. The IL-21 mutein may comprise an amino acid sequence of any of SEQ ID NOs: 159, 161, 238, 241, 242, or 244. The IL-21 mutein may be directly attached to the anti-PD-1 antibody and does not comprise a peptide linker.

[0141] The fusion protein may comprise two IL-21 muteins, each of which is directly attached to the C-terminus of a heavy chain of the anti-PD-1 antibody and each of which have the same amino acid sequence. The IL-21 mutein may comprise an amino acid substitution listed in Table 4 or a sequence of a SEQ ID NO: listed in Table 4. The IL-21 mutein may comprise an amino acid substitution listed in Table 5 or a sequence of a SEQ ID NO: listed in Table 5. The IL-21 mutein may comprise the amino acid substitutions listed in Table 7 or a sequence of a SEQ ID NO: listed in Table 7. The IL-21 mutein may comprise the amino acid substitutions listed in any one of Tables 6 and 8-14 or a sequence of a SEQ ID NO: listed in these Tables. The IL-21 mutein may comprise an amino acid sequence of any of SEQ ID NOs: 159, 161, 237, 238, 241, and 244. The IL-21 mutein may be directly attached to the anti-PD-1 antibody and does not comprise a peptide linker.

[0142] The fusion protein may comprise an amino acid sequence of an antibody constant region described herein fused to an amino acid sequence of any IL-21 mutein described herein. The fusion protein may comprise an amino acid sequence of an antibody constant region described herein, which is not glycosylated, fused to an amino acid sequence of any IL-21 mutein described herein. The fusion protein may comprise a constant region comprising an amino acid sequence of any one of SEQ ID NOs: 265-267, 282, 284-311, 472-495, and 544-555, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to any one of SEQ ID NOs: 265-267, 282, 284-311, 472-495, and 544-555, fused to an IL-21 mutein comprising any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283. The fusion protein may comprise an amino acid sequence of any one of SEQ ID NOs: 268-281, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 268-281.

[0143] The fusion protein may comprise a construct as described in Figure 4A, 4B, or 4C. The fusion protein may comprise (i) an anti-PD-1 antibody (or antigen binding antibody fragment thereof) described herein; and (ii) an IL-21 mutein described herein. The fusion protein may comprise (i) an anti-PD-1 antibody (or antigen binding antibody fragment thereof) described herein; (ii) a charge pair mutation described herein; and (iii) an IL-21 mutein described herein (see, e.g., Figure 4C). The fusion protein may comprise (i) an anti-PD-1 antibody (or antigen binding antibody fragment thereof) described herein, wherein the heavy chain sequences of said anti-PD-1 antibody (or antigen binding antibody fragment thereof) do not comprise a C-terminal lysine; (ii) a charge pair mutation described herein; and (iii) an IL-21 mutein described herein.

[0144] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372, and 382, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) an HC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 314, 324, 334, 344, 3545, 364, 374, and 384, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 315, 325, 335, 345, 355, 365, 375, and 385, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (e) an LC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 316, 326, 336, 346, 356, 366, 376, and 386, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (f) an LC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 317, 327, 337, 347, 357, 367, 377, and 387, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table D and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table D. The antigen-binding protein may comprise 3, 4, 5, or 6 of the amino acid sequences designated by the SEQ ID NOs: in a single column of Table D. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 312-317; (b) SEQ ID NOs: 322-327; (c) SEQ ID NOs: 332-337; (d) SEQ ID NOs: 342-347; (e) SEQ ID NOs: 352-357; (f) SEQ ID NOs: 362-367; (g) SEQ ID NOs: 372-377; and (h) SEQ ID NOs: 382-387. The anti-PD-1 antibody protein product may comprise such regions.

[0145] The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise (a) a heavy chain variable region amino acid sequence set forth in in Table E or a sequence selected from the group consisting of: 318, 328, 338, 348, 358, 368, 378, and 388, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (b) a light chain variable region amino acid sequence set forth in Table E or a sequence selected from the group consisting of: 319, 329, 339, 349, 359, 369, 379, and 389, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (c) both (a) and (b). The anti-PD-1 antibody (or antigen binding antibody fragment thereof) may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389. The antibody constant region may comprise an amino acid sequence of any one of SEQ ID NOs: 265-267, 282, and 284-311, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to any one of SEQ ID NOs: 265-267, 282, and 284-311, fused to an IL-21 mutein comprising any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283. The antigen-binding protein may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391. The fusion protein may comprise (I) a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391, and (II) an IL-21 mutein comprising any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283.

[0146] The fusion protein may comprise a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 361 and 562 or SEQ ID NOs: 361 and 563. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 562). The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 563).

[0147] The fusion protein may comprise a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 361 and 564 or SEQ ID NOs: 361 and 565. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 564). The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 565).

[0148] The fusion protein may comprise a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations and a linker (e.g., SEQ ID NO: 267), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 361 and 566. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 566).

[0149] The fusion protein may comprise a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations and a linker (e.g., SEQ ID NO: 267), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 361 and 567. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 567).

[0150] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V1 charge pair mutations (e.g., SEQ ID NOs: 306 and 307 or SEQ ID NOs: 308 and 309), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising any one of SEQ ID NOs: 309), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 361 and 568. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 568 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 574).

[0151] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V103 charge pair mutations (e.g., SEQ ID NOs: 484 and 485 or SEQ ID NOs: 486 and 487), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V103 charge pair mutations (e.g.i.e., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 487), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 361 and 569. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 569 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 575).

[0152] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V131 charge pair mutations (e.g., SEQ ID NOs: 490 and 491 or SEQ ID NOs: 492 and 493), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 493), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 361 and 570. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 570 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 576).

[0153] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V1 charge pair mutations (e.g., SEQ ID NOs: 306 and 307 or SEQ ID NOs: 308 and 309), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 309), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 361 and 571. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 571 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 574).

[0154] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V103 charge pair mutations (e.g., SEQ ID NOs: 484 and 485 or SEQ ID NOs: 486 and 487), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 487), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 361 and 572. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 572 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 575).

[0155] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V131 charge pair mutations (e.g., SEQ ID NOs: 490 and 491 or SEQ ID NOs: 492 and 493), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E357K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 493), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 358 and a light chain variable region of SEQ ID NO: 359. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 361 and 573. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 573 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 576).

[0156] The fusion protein may comprise a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 389 and 496 or SEQ ID NOs: 389 and 519. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 496). The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 519).

[0157] The fusion protein may comprise a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 389 and 497 or SEQ ID NOs: 389 and 498. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and the fused heavy chain-IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 497). The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 498).

[0158] The fusion protein may comprise a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations and a linker (e.g., SEQ ID NO: 267), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 389 and 499. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 499).

[0159] The fusion protein may comprise a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations and a linker (e.g., SEQ ID NO: 267), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 389 and 500. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 500).

[0160] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V1 charge pair mutations (e.g., SEQ ID NOs: 306 and 307 or SEQ ID NOs: 308 and 309), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 309), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 389 and 501. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 501, and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 556).

[0161] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V103 charge pair mutations (e.g., SEQ ID NOs: 484 and 485 or SEQ ID NOs: 486 and 487), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 487), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 389 and 502. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 502 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 557).

[0162] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V131 charge pair mutations (e.g., SEQ ID NOs: 490 and 491 or SEQ ID NOs: 492 and 493), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 493), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 389 and 503. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 503 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 558).

[0163] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V1 charge pair mutations (e.g., SEQ ID NOs: 306 and 307 or SEQ ID NOs: 308 and 309), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 309), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 389 and 504. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 504 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 556).

[0164] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V103 charge pair mutations (e.g., SEQ ID NOs: 484 and 485 or SEQ ID NOs: 486 and 487), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 487), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 389 and 505. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 505 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 557).

[0165] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V131 charge pair mutations (e.g., SEQ ID NOs: 490 and 491 or SEQ ID NOs: 492 and 493), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising any one of SEQ ID NOs: 493), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 388 and a light chain variable region of SEQ ID NO: 389. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 389 and 506. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 506 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 558).

[0166] The fusion protein may comprise a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 369 and 507 or SEQ ID NOs: 369 and 508. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 507). The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 508).

[0167] The fusion protein may comprise a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 369 and 509 or SEQ ID NOs: 369 and 510. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 509). The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 510).

[0168] The fusion protein may comprise a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations and a linker (e.g., SEQ ID NO: 267), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 369 and 511. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 511).

[0169] The fusion protein may comprise a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and each heavy chain comprises a constant region sequence comprising SEFL2-2 mutations and a linker (e.g., SEQ ID NO: 267), wherein each of the two IL-21 muteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a homodimer comprising the amino acid sequences of SEQ ID NOs: 369 and 512. The fusion protein may comprise a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 512).

[0170] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V1 charge pair mutations (e.g., SEQ ID NOs: 306 and 307 or SEQ ID NOs: 308 and 309), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising any one of SEQ ID NOs: 309), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 369 and 513. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 513 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 559).

[0171] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V103 charge pair mutations (e.g., SEQ ID NOs: 484 and 485 or SEQ ID NOs: 486 and 487), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 487), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 369 and 514. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 514 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 560).

[0172] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V131 charge pair mutations (e.g., SEQ ID NOs: 490 and 491 or SEQ ID NOs: 492 and 493), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 493), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76A (i.e., comprises the sequence of SEQ ID NO: 244). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 369 and 515. In exemplary aspects, the fusion protein comprises a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 515 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 561).

[0173] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V1 charge pair mutations (e.g., SEQ ID NOs: 306 and 307 or SEQ ID NOs: 308 and 309), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 309), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 369 and 516. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 516 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 559).

[0174] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V103 charge pair mutations (e.g., SEQ ID NOs: 484 and 485 or SEQ ID NOs: 486 and 487), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 487), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 369 and 517. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 517 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 560).

[0175] The fusion protein may comprise an IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a pair of heavy chains comprising constant region sequences comprising SEFL2-2 mutations and V131 charge pair mutations (e.g., SEQ ID NOs: 490 and 491 or SEQ ID NOs: 492 and 493), wherein the IL-21 mutein monomer is attached to the heavy chain which contains the E357K and D399K V1 charge pair mutations (e.g., the IL-21 mutein monomer is attached to a heavy chain comprising SEQ ID NOs: 493), and wherein the IL-21 mutein comprises amino acid substitutions R9E and R76E (i.e., comprises the sequence of SEQ ID NO: 245). The anti-PD-1 antibody may comprise a heavy chain variable region of SEQ ID NO: 368 and a light chain variable region of SEQ ID NO: 369. The anti-PD-1 antibody may comprise the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. The fusion protein may comprise a monomer comprising the amino acid sequences of SEQ ID NOs: 369 and 518. The fusion protein may comprise a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 518 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 561).

[0176] The fusion protein may comprise an IL-21 mutein homodimer as shown in Figure 4A or 4B, or IL-21 mutein monomer as shown in Figure 4C, and an anti-PD-1 antibody comprising the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a heavy chain constant region sequence comprising any one of SEQ ID NOs 544-555. The fusion protein may comprise an IL-21 mutein homodimer as shown in Figure 4A or 4B, or IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20A2.003 (SEQ ID NOs: 365-367), the three heavy chain CDRs of antibody 20A2.003 (SEQ ID NOs: 362-364), and a heavy chain constant region sequence of SEQ ID NO: 525 or 527.

[0177] The fusion protein may comprise an IL-21 mutein homodimer as shown in Figure 4A or 4B, or IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a heavy chain constant region sequence comprising any one of SEQ ID NOs 544-555. The fusion protein may comprise an IL-21 mutein homodimer as shown in Figure 4A or 4B, or IL-21 mutein monomer as shown in Figure 4, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 22D4.017 (SEQ ID NOs: 385-387), the three heavy chain CDRs of antibody 22D4.017 (SEQ ID NOs: 382-384), and a heavy chain constant region sequence of SEQ ID NO 529 or 531.

[0178] The fusion protein may comprise an IL-21 mutein homodimer as shown in Figure 4A or 4B, or IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a heavy chain constant region sequence comprising any one of SEQ ID NOs 544-555. The fusion protein may comprise an IL-21 mutein homodimer as shown in Figure 4A or 4B, or IL-21 mutein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises the three light chain CDRs of antibody 20C1.009 (SEQ ID NOs: 355-357), the three heavy chain CDRs of antibody 20C1.009 (SEQ ID NOs: 352-354), and a heavy chain constant region sequence of SEQ ID NO 521 or 523.Antigen-Binding Proteins

[0179] Described herein are PD-1 antigen binding proteins. The PD-1 antigen-binding protein may be an anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product described herein. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372, and 382, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) an HC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 314, 324, 334, 344, 3545, 364, 374, and 384, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 315, 325, 335, 345, 355, 365, 375, and 385, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (e) an LC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 316, 326, 336, 346, 356, 366, 376, and 386, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (f) an LC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 317, 327, 337, 347, 357, 367, 377, and 387, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table D and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table D. The antigen-binding protein may comprise at least 3, 4, or 5 of the amino acid sequences designated by the SEQ ID NOs: in a single column of Table D.

[0180] The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 312-317; (b) SEQ ID NOs: 322-327; (c) SEQ ID NOs: 332-337; (d) SEQ ID NOs: 342-347; (e) SEQ ID NOs: 352-357; (f) SEQ ID NOs: 362-367; (g) SEQ ID NOs: 372-377; and (h) SEQ ID NOs: 382-387. The amino acid sequences of Table D may be separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s). The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise (a) a heavy chain variable region amino acid sequence set forth in in Table E or a sequence selected from the group consisting of: 318, 328, 338, 348, 358, 368, 378, and 388, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (b) a light chain variable region amino acid sequence set forth in Table E or a sequence selected from the group consisting of: 319, 329, 339, 349, 359, 369, 379, and 389, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (c) both (a) and (b). The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389. The anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product may comprise (I) a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389 and (II) a constant region comprising any one of SEQ ID NOs: 265-267, 282, 284-311, 472-495, and 544-555. The antigen-binding protein may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391. The antigen-binding protein may comprise an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to one or more of the SEQ ID NOs: above.

[0181] The antigen binding protein described above may be an anti-PD-1 antibody, or an antigen binding antibody fragment thereof.

[0182] Additionally described are conjugates comprising a PD-1 antigen binding protein described herein and a heterologous moiety. The heterologous moiety may be any molecule which is different from the PD-1 antigen-binding protein described herein. The heterologous moiety may be a heterologous peptide or polypeptide, a targeting agent, a diagnostic label, a polymer, a nucleic acid, a quantum dot, a small molecule, a toxin, a carbohydrate, an amino acid, or other therapeutic or diagnostic agent. The heterologous moiety may be an IL-21 mutein as described herein.

[0183] Additionally described are fusion proteins comprising a PD-1 antigen binding protein described herein and a heterologous polypeptide or peptide. The heterologous polypeptide may be an IL-21 mutein as described herein.Methods of Making Antibodies

[0184] Suitable methods of making antibodies, antigen binding antibody fragments, and antibody protein products are known in the art. For instance, standard hybridoma methods for producing antibodies are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). An exemplary method of preparing anti-PD-1 monoclonal antibodies or the present disclosure is provided herein in EXAMPLES.

[0185] Depending on the host species, various adjuvants can be used to increase the immunological response leading to greater antibody production by the host. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.

[0186] Other methods of antibody production are summarized in Table F. TABLE FTechnique Exemplary references EBV-hybridoma methods and Bacteriophage vector expression systemsHaskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), Roder et al., Methods Enzymol., 121, 140-67 (1986), and Huse et al., Science, 246, 1275-81 (1989)).methods of producing antibodies in non-human animalsU.S. Patents 5,545,806, U.S. 5,569,825, and U.S. 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagentsOrlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G and Milstein C (Nature 349: 293-299, 1991).methods of producing recombinant proteinsProtein production and purification" Nat Methods 5(2): 135-146 (2008).Phage displayJaneway et al., supra, Huse et al., supra, and U.S. Patent 6,265,150). Related methods also are described in U.S. Patent No. 5,403,484; U.S. Patent No. 5,571,698; U.S. Patent No. 5,837,500; U.S. Patent No. 5,702,892. The techniques described in U.S. Patent No. 5,780,279; U.S. Patent No. 5,821,047; U.S. Patent No. 5,824,520; U.S. Patent No.5,855,885; U.S. Patent No. 5,858,657; U.S. Patent No. 5,871,907; U.S. Patent No. 5,969,108; U.S. Patent No. 6,057,098; and U.S. Patent No. 6,225,447Antibodies can be produced by transgenic miceU.S. Patent Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.

[0187] Methods of testing antibodies for the ability to bind to PD-1 regardless of how the antibodies are produced are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266, and the above section relating to competition assays). Other binding assays, e.g., competitive binding assays or competition assays, which test the ability of an antibody to compete with a second antibody for binding to an antigen, or to an epitope thereof, are known in the art and can be used to test the ability of an antibody to bind to PD-1. See, e.g., U.S. Patent Application Publication No. US20140178905, Chand et al., Biologicals 46: 168-171 (2017); Liu et al., Anal Biochem 525: 89-91 (2017); and Goolia et al., J Vet Diagn Invest 29(2): 250-253 (2017). Also, other methods of comparing two antibodies are known in the art, and include, for example, surface plasmon resonance (SPR). SPR can be used to determine the binding constants of the antibody and second antibody and the two binding constants can be compared.Heterologous moieties: polymers, carbohydrates, lipids and therapeutic agents

[0188] The conjugate as described herein may comprise an IL-21 mutein linked to a polymer. The polymer may be selected from the group consisting of: polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt, polypropylene, polyethylenes including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene. In specific embodiments, the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).

[0189] The conjugate as described herein may comprise an IL-21 mutein linked to a carbohydrate. The carbohydrate may be a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (e.g., starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, or galactomannan).

[0190] The heterologous moiety may be a lipid. The lipidmay be a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g., mono-, di-, trisubstituted glycerols), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or a polyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, a phospholipid.

[0191] The conjugate as described herein may comprise an IL-21 mutein linked to a therapeutic agent. The therapeutic agent can be any of those known in the art. The therapeutic agent may be an immunotherapy agent insofar as the agent stimulates an immune response. The immunotherapy agent may be cancer vaccine. The immunotherapy agent may be a monoclonal antibody. The immunotherapy agent may be an immune checkpoint inhibitor, e.g., an inhibitor of CTLA4, PD-1, PD-L1. The monoclonal antibody may be specific for a protein in an immune-checkpoint pathway. The protein of the immune-checkpoint pathway can be, for example, CTLA4, PD-1, PD-L1, B7-H3, B7H4, or TIM3. For instance, the antigen-binding proteins of the present disclosure can be conjugated to atezolizumab, avelumab, ipilimumab, tremelimumab, BMS-936558, MK3475, CT-011, AM-224, MDX-1105, IMP321, MGA271.

[0192] The therapeutic agent may be a cytokine, lymphokine, growth factor, or hematopoietic factor effective in inhibiting tumor metastasis and / or having an antiproliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to: M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Additional growth factors for use herein include angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor α, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2 α, cytokine-induced neutrophil chemotactic factor 2 β, β endothelial cell growth factor, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neutrophic factor receptor α 1, glial cell line-derived neutrophic factor receptor α 2, growth related protein, growth related protein α, growth related protein β, growth related protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof. In exemplary embodiments, the therapeutic agent comprises an antibody specific for any one of the aforementioned cytokines, lymphokines, growth factors, or other hematopoietic factors.Nucleic acids

[0193] Further described are nucleic acids comprising a nucleotide sequence encoding an IL-21 mutein as described herein, a conjugate comprising an IL-21 mutein, or a fusion protein comprising an IL-21 mutein. For example, the nucleic acid may comprise a nucleotide sequence encoding a heavy chain of an anti-PD-1 antibody followed by a nucleotide sequence encoding an IL-21 mutein of the present disclosure. The nucleotide sequence encoding a heavy chain and the nucleotide sequence encoding the IL-21 mutein may flank a nucleotide sequence encoding a peptide linker comprising the amino acid sequence of GGGGS (SEQ ID NO: 262). Alternatively, the nucleic acid may not comprise a nucleotide sequence encoding a peptide linker and the nucleotide sequence encoding a heavy chain of an anti-PD-1 antibody is tandem to the nucleotide sequence encoding an IL-21 mutein as described herein.

[0194] The nucleic acid may comprise a nucleotide sequence encoding an IL-21 mutein comprising an amino acid sequence of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to an amino acid sequence of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283.

[0195] The nucleic acid may comprise a nucleotide sequence encoding a peptide linker of SEQ ID NO: 262 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 262.

[0196] The nucleic acid may comprise a nucleotide sequence encoding a fusion protein comprising an amino acid sequence of an antibody constant region described herein fused to an amino acid sequence of any IL-21 mutein described herein. The nucleic acid may comprise a nucleotide sequence encoding a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 265-267, and 282, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to any one of SEQ ID NOs: 265-267, and 282, fused to any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283. The nucleic acid may comprise a nucleotide sequence encoding a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 268-281, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 268-281.

[0197] The nucleic acid may comprise a nucleotide sequence encoding an anti-PD-1 antibody comprising a heavy chain constant region amino acid sequence of any one of SEQ ID NOs: 265-267, 282, 284-311, 472-495 and 544-555, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to any one of SEQ ID NO: 265-267, 282, 284-311, 472-495 and 544-555.

[0198] Further described are nucleic acids comprising a nucleotide sequence encoding a PD-1 antigen binding protein of the present disclosure. The nucleotide sequence may comprise a sequence encoding a heavy chain CDR or light chain CDR, a heavy chain variable region or light chain variable region or heavy chain sequence or light chain sequence. See Table G below. The nucleotide sequence may comprise any one of SEQ ID NOs: 392-471. Further described are pairs of nucleotide sequences comprising (a) SEQ ID NOs: 398 and 399, (b) SEQ ID NOs: 408 and 409, (c) SEQ ID NOs: 418 and 419, (d) SEQ ID NOs: 428 and 429, (e) SEQ ID NOs: 438 and 439, (f) SEQ ID NOs: 448 and 449, (g) SEQ ID NOs: 458 and 459, or (h) SEQ ID NOs: 468 and 469. Additionally described are pairs of nucleotide sequences comprising (a) SEQ ID NOs: 400 and 401, (b) SEQ ID NOs: 410 and 411, (c) SEQ ID NOs: 420 and 421, (d) SEQ ID NOs: 430 and 431, (e) SEQ ID NOs: 440 and 441, (f) SEQ ID NOs: 450 and 451, (g) SEQ ID NOs: 460 and 461, or (h) SEQ ID NOs: 470 and 471. TABLE G20A2 20C1 22D4 20C1.006 20C1.009 20A2.003 22D4.006 22D4.017 HC CDR1392402412422432442452462HC CDR2393403413423433443453463HC CDR3394404414424434444454464LC CDR1395405415425435445455465LC CDR2396406416426436446456466LC CDR3397407417427437447457467HC VARIABLE398408418428438448458468LC VARIABLE399409419429439449459469HC FULL LENGTH400410420430440450460470LC FULL LENGTH401411421431441451461471

[0199] The nucleic acid molecule may comprise a nucleotide sequence encoding a conjugate or fusion protein of the present disclosure. By "nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, or modified forms thereof, which can be single-stranded or double- stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered inter-nucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. The nucleic acid can comprise any nucleotide sequence which encodes any of the antigen-binding proteins or polypeptides of the present disclosure. The nucleic acid may not comprise any insertions, deletions, inversions, and / or substitutions. The nucleic acid may comprise one or more insertions, deletions, inversions, and / or substitutions.

[0200] The nucleic acids as described herein may be recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0201] The nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra; and Ausubel et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chIorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridme, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6< -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N -substituted adenine, 7-methylguanine, 5-methylammomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6< -isopentenyladenine, uracil- 5-oxyacetic acid (v), wybutoxosine, pseudouratil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3- (3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present disclosure can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).Vectors

[0202] The nucleic acids as described herein may be incorporated into a vector. In this regard, described herein are vectors comprising any of the presently disclosed nucleic acids. The vector may be a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors as described herein are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The presently described vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. The altered nucleotides or non-naturally occurring internucleotide linkages may not hinder the transcription or replication of the vector.

[0203] The vector as described herein can be any suitable vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJoIIa, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGTIO, λGTl 1, λZapII (Stratagene), λEMBL4, and λNMl 149, also can be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The vector may be a viral vector, e.g., a retroviral vector.

[0204] The vectors as described herein can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from CoIEl, 2 µ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0205] The vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0206] The vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the presently disclosed expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0207] The vector can comprise a native or normative promoter operably linked to the nucleotide sequence encoding the polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the IL-21, conjugate, or fusion protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.Host cells

[0208] Decsribed herein are host cells comprising a nucleic acid or vector as described herein. As used herein, the term "host cell" refers to any type of cell that can contain the presently disclosed vector and is capable of producing an expression product encoded by the nucleic acid (e.g., mRNA, protein). The host cell may be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. The host cell may be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage.

[0209] The cell may be a eukaryotic cell, including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell. Such host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4: 217 (2013). The eukaryotic cells may be mammalian cells. The mammalian cells may be non-human mammalian cells. The cells may be Chinese Hamster Ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3, CS9), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to be deficient in dihydrofolatereductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryo fibroblast cells NIH 3T3, mouse fibroblast cells L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2): 257-263 (2013)). In a particular embodiment, the host cell is CS9 (a CHO cell line).

[0210] For purposes of amplifying or replicating the vector, the host cell may be a prokaryotic cell, e.g., a bacterial cell.

[0211] Also described herein is a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising the host cell comprising vectors described, in addition to at least one other cell, which does not comprise any of the vectors.

[0212] Alternatively, the population of cells may be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the vector. The population may be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a vector, such that all cells of the population comprise the vector. The population of cells may be a clonal population comprising host cells comprising a vector as described herein.Pharmaceutical Compositions

[0213] Compositions comprising an IL-21 mutein, a conjugate comprising the IL-21 mutein, a fusion protein comprising the IL-21 mutein and a polypeptide, a PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate comprising the PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a fusion protein comprising the PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a nucleic acid, vector, or host cell, as described herein, or a combination thereof, are described herein. The compositions may comprise the IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof, in isolated and / or purified form. The composition may comprise a single type (e.g., structure) of an IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate, fusion protein, nucleic acid, vector, or host cell as described herein, or comprises a combination of two or more different types (e.g., different structures) of IL-21 muteins, PD-1 antigen-binding proteins, conjugates, fusion proteins, nucleic acids, vectors or host cells as described herein.

[0214] The composition may comprise agents which enhance the chemico-physico features of the IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate, fusion protein, nucleic acid, vector, or host cell, e.g., via stabilizing, for example, the IL-21 mutein or fusion protein at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation, e.g., oxidation protease mediated degradation, increasing half-life of, for example, the IL-21 mutein or fusion protein, etc. The composition may comprise any of the agents disclosed herein as a heterologous moiety or conjugate moiety, optionally, in admixture with the IL-21 muteins, conjugates, fusion proteins, nucleic acids, vectors, or host cells as described herein.

[0215] The composition may additionally comprise a pharmaceutically acceptable carrier, diluents, or excipient. The IL-21 muteins, PD-1 antigen-binding proteins (e.g., an anti-PD-1 antibodies), conjugates, fusion proteins, nucleic acids, vectors, or host cells as presently described (hereinafter referred to as "active agents") may be formulated into a pharmaceutical composition comprising the active agent, along with a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, further described are pharmaceutical compositions comprising an active agent (i.e., any of the IL-21 muteins, PD-1 antigen-binding proteins (e.g., an anti-PD-1 antibodies), conjugates, fusion proteins, nucleic acids, vectors, or host cells of the present disclosure), which pharmaceutical composition may be intended for administration to a subject, e.g., a mammal.

[0216] The active agent may be present in the pharmaceutical composition at a purity level suitable for administration to a patient. The active agent may have a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, and a pharmaceutically acceptable diluent, carrier or excipient. The compositions may contain an active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0217] The pharmaceutical compositions may comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

[0218] The pharmaceutical composition can comprise any pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000). Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980).

[0219] The pharmaceutical composition may comprise formulation materials that are nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions may comprise an active agent and one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents. The pharmaceutical composition may comprise one or more polyols and / or one or more surfactants, optionally, in addition to one or more excipients, including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.

[0220] The pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as bcnzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbatc, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (A. R. Genrmo, ed.), 1990, Mack Publishing Company.

[0221] The pharmaceutical compositions can be formulated to achieve a physiologically compatible pH. The pH of the pharmaceutical composition can be for example between about 4 or about 5 and about 8.0 or about 4.5 and about 7.5 or about 5.0 to about 7.5. The pH of the pharmaceutical composition may be between 5.5 and 7.5.Routes of Administration

[0222] With regard to the present disclosure, the active agent, or pharmaceutical composition comprising the same, can be administered to the subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion on routes of administration is merely provided to illustrate exemplary embodiments and should not be construed as limiting the scope in any way.

[0223] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term, "parenteral" means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agent of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2- dimethyl-153-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0224] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0225] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminoproplonates, and 2-alkyl -imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0226] The parenteral formulations may contain from about 0.5% to about 25% by weight of the active agent of the present disclosure in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions can contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0227] Injectable formulations are in accordance with the present disclosure. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).Dosages

[0228] The active agents as described herein are believed to be useful in methods of inhibiting a PD-1 signaling, while providing IL-21 signaling, as described herein, and are thus believed to be useful in methods of treating or preventing one or more diseases, e.g., cancer. The amount or dose of the active agent administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the active agent of the present disclosure should be sufficient to treat cancer as described herein in a period of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks or longer, e.g., 5 to 20 or more weeks, from the time of administration. The time period could be even longer. The dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0229] Many assays for determining an administered dose are known in the art. For purposes herein, an assay, which comprises comparing the extent to which cancer is treated upon administration of a given dose of the active agent of the present disclosure to a mammal among a set of mammals, each set of which is given a different dose of the active agent, could be used to determine a starting dose to be administered to a mammal. The extent to which cancer is treated upon administration of a certain dose can be represented by, for example, the cytotoxicity of the active agent or the extent of tumor regression achieved with the active agent in a mouse xenograft model. Methods of measuring cytotoxicity of the fusion proteins and methods of assaying tumor regression are known in the art.

[0230] The dose of the active agent of the present disclosure also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular active agent of the present disclosure. Typically, the attending physician will decide the dosage of the active agent of the present disclosure with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, active agent of the present disclosure to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the present disclosure, the dose of the active agent of the present disclosure can be about 0.0001 to about 1 g / kg body weight of the subject being treated / day, from about 0.0001 to about 0.001 g / kg body weight / day, or about 0.01 mg to about 1 g / kg body weight / day.Controlled Release Formulations

[0231] The active agents described herein can be modified into a depot form, such that the manner in which the active agent as described herein is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of active agents as described herein can be, for example, an implantable composition comprising the active agents and a porous or non-porous material, such as a polymer, wherein the active agent is encapsulated by or diffused throughout the material and / or degradation of the non-porous material. The depot is then implanted into the desired location within the body of the subject and the active agent is released from the implant at a predetermined rate.

[0232] The pharmaceutical composition comprising the active agent may be modified to have any type of in vivo release profile. The pharmaceutical composition may be an immediate release, controlled release, sustained release, extended release, delayed release, or bi-phasic release formulation. Methods of formulating peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009) and International Patent Application Publication Nos. WO 2008 / 130158, WO2004 / 033036; WO2000 / 032218; and WO 1999 / 040942.

[0233] The instant compositions can further comprise, for example, micelles or liposomes, or some other encapsulated form, or can be administered in an extended release form to provide a prolonged storage and / or delivery effect.Combinations

[0234] The fusion proteins or antigen-binding proteins (e.g., anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product) described herein may be administered alone, and alternatively, may be administered in combination with another therapeutic agent, e.g., another active agent of different type (e.g., structure). The other therapeutic may aim to treat or prevent cancer. The other therapeutic may be a chemotherapeutic agent. The other therapeutic may be an agent used in radiation therapy for the treatment of cancer. Accordingly, the fusion proteins or antigen-binding proteins (e.g., anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product) described herein may be administered in combination with one or more of platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides. An IL-21 fusion protein described herein (e.g., an anti-PD-1 antibody fused to an IL-21 mutein) may be combined with an antigen-binding protein (e.g., anti-PD-1 antibody, antigen binding antibody fragment thereof, or anti-PD-1 antibody protein product).

[0235] Any of antibodies 20A2, 20C1, 22D4, 20C1.006, 20C1.009, 20A2.003, 22D4.006, 22D4.017 may be administered in combination with an IL-21 fusion protein described herein including, for example: a fusion protein comprising a homodimer or monomer selected from: a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 496); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and the fused heavy chain-IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 497); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 498); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 499); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 500); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 501 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 555); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 502 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 556); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 503 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 557); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 504 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 555); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 505 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 556); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 506 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 557); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 507); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 508); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 509); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 510); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 511); a homodimer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each of which is fused to an IL-21 mutein, and each heavy chain-IL-21 mutein fusion comprises the amino acid sequence of SEQ ID NO: 512); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 513 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 558); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 514 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 559); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 515 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 560); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 516 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 558); a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 517 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 559); or a monomer comprising two antibody light chains (each comprising the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one of which is fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 518 and one of which is not fused to an IL-21 mutein and comprises the amino acid sequence of SEQ ID NO: 560). Kits

[0236] Additionally described are kits comprising an IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibodies), a conjugate, fusion protein, nucleic acid, vector, or host cell as described herein, or a combination thereof. The kit may comprise at least one IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibodies), a conjugate, fusion protein, nucleic acid, vector, or host cell as described herein, or a combination thereof, in a container. The at least one IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibodies), a conjugate, fusion protein, nucleic acid, vector, or host cell as described herein, may be provided in the kit as a unit dose. For purposes herein "unit dose" refers to a discrete amount dispersed in a suitable carrier. The unit dose may be the amount sufficient to provide a subject with a desired effect, e.g., treatment of cancer. The kit may comprise several unit doses, e.g., a week or month supply of unit doses, optionally, each of which is individually packaged or otherwise separated from other unit doses. The components of the kit / unit dose may be packaged with instructions for administration to a patient. The kit may comprise one or more devices for administration to a patient, e.g., a needle and syringe, and the like. The at least one IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibodies), a conjugate, fusion protein, nucleic acid, vector, or host cell as described herein, or a combination thereof, may be pre-packaged in a ready to use form, e.g., a syringe, an intravenous bag, etc. The ready to use form may be for a single use. Te kit may comprise multiple single use, ready to use forms of the at least one IL-21 mutein, PD-1 antigen-binding protein (e.g., an anti-PD-1 antibodies), a conjugate, fusion protein, nucleic acid, vector, or host cell as described herein. The kit may further comprise other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein.Methods of Manufacture

[0237] The IL-21 muteins as described herein may be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides are described in, for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Additional exemplary methods of making the peptides described herein are set forth herein.

[0238] The IL-21 muteins described herein may be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), Multiple Peptide Systems (San Diego, CA), Peptide 2.0 Inc. (Chantilly, VA), and American Peptide Co. (Sunnyvale, CA). In this respect, the IL-21 muteins can be synthetic, recombinant, isolated, and / or purified.

[0239] Also, the IL-21 muteins may be recombinantly produced using a nucleic acid encoding the amino acid sequence of the peptide using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0240] Methods of making an IL-21 mutein are described herein. The method may comprise culturing a host cell as described herein to express the IL-21 mutein and harvesting the expressed IL-21 mutein.

[0241] Methods of making fusion protein comprising an IL-21 mutein are also described herein. The method may comprise culturing a host cell as described herein to express the fusion protein and harvesting the expressed fusion protein.

[0242] The method may comprise culturing a host cell comprising a nucleic acid encoding the IL-21 mutein or fusion protein as described herein so as to express the IL-21 mutein or fusion protein. The host cell can be any of the host cells described herein. The host cell may be selected from the group consisting of: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. The step of culturing a host cell may comprise culturing the host cell in a growth medium to support the growth and expansion of the host cell. The growth medium may increase cell density, culture viability and productivity in a timely manner. The growth medium may comprise amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. The growth medium may be a fully chemically defined media consisting of amino acids, vitamins, trace elements, inorganic salts, lipids and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also helps maintain pH and osmolality. Several growth media are commercially available and are described in the art. See, e.g., Arora, "Cell Culture Media: A Review" MATER METHODS 3:175 (2013).

[0243] The method of making an IL-21 mutein or fusion protein as described herein may comprise culturing the host cell in a feed medium. The method may comprise culturing in a feed medium in a fed-batch mode. Methods of recombinant protein production are known in the art. See, e.g., Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5): 466-477 (2010).

[0244] The method making an IL-21 mutein or fusion protein can comprise one or more steps for purifying the mutein or protein from a cell culture or the supernatant thereof and preferably recovering the purified protein. The method may comprise one or more chromatography steps, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. The method may comprise purifying the protein using a Protein A affinity chromatography resin.

[0245] The method may further comprise steps for formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.Methods of Use

[0246] Methods of treatment are additionally described herein. The method may be a method of treating a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present disclosure in an amount effective to treat the subject.

[0247] The pharmaceutical compositions of the present disclosure are useful for inhibiting PD-1 signaling and / or activating IL-21 signaling. Without being bound to a particular theory, [1] the PD-1 inhibiting activity of the compositions provided herein allow such entities to be useful in methods of enhancing T cell activity and enhancing an immune response, and, in particular, an immune response against a tumor or cancer; and / or [2] the IL-21 activating activity of the compositions provided herein allow such entities to enhance T cell survival and effector function, restrict terminal differentiation and loss of replicative potential, promote T cell longevity by shifting activated effector cells towards a more naïve T cell phenotype (e.g., by enhancing CCR7 expression) , and enhance cytotoxicity against target (e.g., cancer) cell (e.g., by increasing IFNγ and granzyme B production.

[0248] Accordingly, described herein are methods of enhancing T cell activity in a subject, enhancing T cell survival and effector function, restricting terminal differentiation and loss of replicative potential, promoting T cell longevity, and enhancing cytotoxicity against target (e.g., cancer) cells. The methods may comprise administering to the subject the pharmaceutical composition of the present disclosure in an effective amount. The T cell activity or immune response may be directed against a cancer cell or cancer tissue or a tumor cell or tumor. The immune response may be a humoral immune response. The immune response may be an innate immune response. The immune response which is enhanced may be a T-cell mediated immune response.

[0249] As used herein, the term "enhance" and words stemming therefrom may not be a 100% or complete enhancement or increase. Rather, there are varying degrees of enhancement of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the pharmaceutical compositions as described herein may enhance, e.g., T cell activity or enhance an immune response, to any amount or level. The enhancement provided by the methods as described herein may be at least or about a 10% enhancement (e.g., at least or about a 20% enhancement, at least or about a 30% enhancement, at least or about a 40% enhancement, at least or about a 50% enhancement, at least or about a 60% enhancement, at least or about a 70% enhancement, at least or about a 80% enhancement, at least or about a 90% enhancement, at least or about a 95% enhancement, at least or about a 98% enhancement).

[0250] Methods of measuring T cell activity and immune responses are known in the art. T cell activity can be measured by, for example, a cytotoxicity assay, such as those described in Fu et al., PLoS ONE 5(7): e11867 (2010). Other T cell activity assays are described in Bercovici et al., Clin Diagn Lab Immunol. 7(6): 859-864 (2000). Methods of measuring immune responses are described in e.g., Macatangay et al., Clin Vaccine Immunol 17(9): 1452-1459 (2010), and Clay et al., Clin Cancer Res.7(5): 1127-35 (2001).

[0251] Additionally described herein are methods of treating a subject with cancer and methods of treating a subject with a solid tumor. The method may comprise administering to the subject the pharmaceutical composition as described herein in an amount effective for treating the cancer or the solid tumor in the subject. The cancer treatable by the methods described herein can be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream. The cancer may be one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In particular aspects, the cancer is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. The tumor may be non-small cell lung cancer (NSCLC), head and neck cancer, renal cancer, triple negative breast cancer, and gastric cancer. In exemplary aspects, the subject has a tumor (e.g., a solid tumor, a hematological malignancy, or a lymphoid malignancy) and the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject. In other exemplary aspects, the tumor is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, renal cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, gastric cancer, lymphoma or leukemia, and the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject.

[0252] As used herein, the term "treat," as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating cancer described herein can provide any amount or any level of treatment. Furthermore, the treatment provided by the method described herein can include treatment of one or more conditions or symptoms or signs of the cancer being treated. Also, the treatment provided by the methods described herein can encompass slowing the progression of the cancer. For example, the methods can treat cancer by virtue of enhancing the T cell activity or an immune response against the cancer, reducing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, and the like. The methods may treat by way of delaying the onset or recurrence of the cancer by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. The methods may treat by way increasing the survival of the subject.Subjects

[0253] The subject may be a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). The mammal may be a human.Exemplary Disclosure

[0254] Described herein is an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 2, QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), wherein "X" represents any amino acid, and wherein the IL-21 mutein amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least 1 amino acid.

[0255] The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 7 amino acidsThe IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5, or 6 amino acids. The IL-21 mutein may comprise an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 1 or 2 amino acids. The difference(s) between the amino acid sequence of the IL-21 mutein and the amino acid sequence of SEQ ID NO: 1 may be within amino acids 10-15, inclusive, or amino acids 105-123, inclusive, of SEQ ID NO: 2, optionally, wherein the difference(s) occur(s) at amino acids 11, 14, 15, 109, 110, 112, 113, 116, 119, 120, and / or 123 of SEQ ID NO: 2. The difference(s) between the amino acid sequence of the IL-21 mutein and the amino acid sequence of SEQ ID NO: 1 may be within amino acids 5-25, inclusive, or amino acids 65-80, inclusive, of SEQ ID NO: 2, optionally, wherein the difference(s) occur(s) at amino acids 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, and / or 80 of SEQ ID NO: 2.

[0256] The IL-21 mutein may comprise an amino acid sequence with one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1). The amino acid substitution may occur at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. The amino acid substitution may occur at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. The IL-21 mutein may comprise an amino acid substitution: a. at position 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an aliphatic amino acid b. at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, wherein the substitute amino acid is an acidic amino acid; c. at position 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1, wherein the substitute amino acid is a basic amino acid; d. at position 5, 8, 9, 70, or 76 of SEQ ID NO: 1, wherein the substitute amino acid is an aromatic amino acid; e. at position 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid comprising a side chain amide; f. at position 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is a non-aromatic amino acid comprising a side chain hydroxyl; g. at position 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, wherein the substitute amino acid is an imino acid; h. at position 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid comprising a sulfur-containing side chain; or i. or a combination thereof.

[0257] The substitute amino acid may be a naturally occurring amino acid. The IL-21 mutein may comprise an amino acid substitution with an amino acid at the position according to TABLE A. Table A is shown below. TABLE AAmino Acid position of SEQ ID NO: 1Amino Acid (in single letter code)Amino Acid position of SEQ ID NO: 1Amino Acid (in single letter code)5A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y72D, G, orP8A, D, E, N, S, T, V, or Y73A, D, E, G, H, I, N, P, Q, S, or V9A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y75D, G, orP11D or S76A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V, orY12A, D, E, N, S, T, or V77D, G, orP13D78D14A, D, or S79D15A, E, I, M, N, Q, S, T, or V80G, or P16D orE109K19D110D23D112D65D, G, orP113K66D, G, orP116A, D, E, I, K, L, M, N, S, T, or V68Q117D69D, G, orP119A, D, E, M, N, Q, S, or T70E, G, P, or Y120D71L123D

[0258] Described herein is an IL-21 mutein comprising an amino acid sequence of any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283.

[0259] The IL-21 mutein may comprise an amino acid sequence with two amino acid substitutions relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1). The amino acid substitution may occur at two of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. The amino acid substitutions may occur at two of positions 5, 9, 15, 70, 71, 72, 73, and 76 of SEQ ID NO: 1. Optionally, the amino acid substitutions occur at two of positions 5, 9, 73, and 76 or SEQ ID NO: 1. One of the substitutions may occur at position 76 of SEQ ID NO: 1. The substitute amino acid at position 76 of SEQ ID NO: 1 may be an aliphatic amino acid or an acidic amino acid. The IL-21 mutein may comprise an amino acid substitution at position 5, 9, or 73 of SEQ ID NO: 1, and the substitute amino acid may be an aliphatic amino acid or acidic amino acid. The IL-21 mutein may comprise an amino acid substitution at position 5 of SEQ ID NO: 1, and the substitute amino acid may be an amino acid with a side chain amide. The aliphatic amino acid may be alanine, the acidic amino acid is glutamic acid, or the amino acid with a side chain amide may be glutamine. Described herein is an IL-21 mutein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 208, 210 to 222, 224 to 248, and 255.

[0260] The IL-21 mutein may bind to the IL-21 receptor with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 receptor. The IL-21 receptor may have an amino acid sequence of SEQ ID NO: 256 or 261. The IL-21 mutein may bind to a IL-21 receptor gamma chain having an amino acid sequence of SEQ ID NO: 257. The IL-21 mutein may bind to the human IL-21 receptor of with a Kd that is greater than or is about 0.04 nM.

[0261] Further described are conjugates. The conjugate may comprise an IL-21 mutein of any one of the preceding paragraphs and a heterologous moiety. The IL-21 may be directly attached to the heterologous moiety. The IL-21 may be attached to the heterologous moiety via a linker. The linker may comprise a peptide, e.g., comprising an amino acid sequence of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 262). The heterologous moiety may be a polypeptide, optionally, wherein the polypeptide is an antigen-binding protein. The heterologous polypeptide may be an antibody or an antigen binding antibody fragment thereof. The antibody may be an anti-PD-1 antibody. The IL-21 mutein may be directly attached to the Fc of the antibody. The IL-21 mutein may be attached to the Fc of the antibody via a linker. The conjugate may comprise a single IL-21 mutein, wherein said single IL-21 mutein is linked to the C-terminus of one of the two antibody heavy chains. The conjugate may comprise two IL-21 muteins, wherein the first IL-21 mutein is linked to the C-terminus of the first antibody heavy chain, and the second IL-21 mutein is linked to the C-terminus of the second antibody heavy chain. Optionally, the first IL-21 has the same amino acid sequence as the second IL-21 mutein. Alternatively, the first IL-21 has a different amino acid sequence than the second IL-21 mutein. The antibody heavy chains may comprise charge pair mutations (e.g., the V1, V4, V103, or V131 mutations). In the conjugate of any one of the preceding paragraphs, the IL-21 mutein may comprise amino acid substitutions at two of positions 5, 9, 73, and 76 of SEQ ID NO: 1. The conjugate may comprise an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 1, except that said IL-21 mutein comprises amino acid substitutions at any two of positions 5, 9, 73, and 76 of SEQ ID NO: 1, and an anti-PD-1 antibody, wherein the IL-21 mutein is linked to the C-terminus of the anti-PD-1 antibody. The conjugate may comprise an anti-PD-1 antibody comprising (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372, and 382, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) an HC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 314, 324, 334, 344, 3545, 364, 374, and 384, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 315, 325, 335, 345, 355, 365, 375, and 385, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (e) an LC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 316, 326, 336, 346, 356, 366, 376, and 386, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (f) an LC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 317, 327, 337, 347, 357, 367, 377, and 387, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). The anti-PD-1 antibody may comprise six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 312-317; (b) SEQ ID NOs: 322-327; (c) SEQ ID NOs: 332-337; (d) SEQ ID NOs: 342-347; (e) SEQ ID NOs: 352-357; (f) SEQ ID NOs: 362-367; (g) SEQ ID NOs: 372-377; and (h) SEQ ID NOs: 382-387. The anti-PD-1 antibody may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389. In exemplary aspects, the anti-PD-1 antibody comprises a constant region comprising an amino acid sequence of any one of SEQ ID NOs: 265-267, 282, 284-311. The anti-PD-1 antibody may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391.

[0262] Also described herein is a fusion polypeptide or fusion protein comprising an IL-21 mutein described herein and a heterologous polypeptide or peptide. The fusion polypeptide or fusion protein may comprise an immunoglobulin or an antigen binding antibody fragment thereof. Also described herein is a nucleic acid comprising a nucleotide sequence encoding an IL-21 mutein described herein. Also described is a vector comprising the nucleic acid described herein. Also described is a host cell comprising the nucleic acid or the vector described herein. Also described is a kit comprising an IL-21 mutein, a nucleic acid, vector, host cell, conjugate, fusion protein, or a combination thereof, as described herein, and a container.

[0263] Also described are pharmaceutical compositions comprising an IL-21 mutein, a nucleic acid, vector, host cell, conjugate, fusion protein, or a combination thereof, as described herein, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0264] Also described are methods of making an IL-21 mutein comprising culturing the host cell as descriebd herein so as to express the IL-21 mutein and harvesting the expressed IL-21 mutein. A method of treating a subject in need thereof is further described. The method comprises administering to the subject in need thereof a pharmaceutical composition of the present disclosure in an amount effective to treat the subject. The subject may have a solid tumor and the pharmaceutical composition may be administered to the subject in an amount effective to treat the solid tumor in the subject. Optionally, the the solid tumor is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma.

[0265] Also described is a PD-1 antigen-binding protein comprising (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372, and 382, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (c) an HC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: SEQ ID NOs: 314, 324, 334, 344, 3545, 364, 374, and 384, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 315, 325, 335, 345, 355, 365, 375, and 385, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (e) an LC CDR2 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 316, 326, 336, 346, 356, 366, 376, and 386, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; (f) an LC CDR3 amino acid sequence set forth in Table D or a sequence selected from the group consisting of: 317, 327, 337, 347, 357, 367, 377, and 387, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). The PD-1 antigen-binding protein may comprise six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 312-317; (b) SEQ ID NOs: 322-327; (c) SEQ ID NOs: 332-337; (d) SEQ ID NOs: 342-347; (e) SEQ ID NOs: 352-357; (f) SEQ ID NOs: 362-367; (g) SEQ ID NOs: 372-377; and (h) SEQ ID NOs: 382-387. The PD-1 antigen-binding protein may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 318 and 319; (b) SEQ ID NOs: 328 and 329; (c) SEQ ID NOs: 338 and 339; (d) SEQ ID NOs: 348 and 349; (e) SEQ ID NOs: 358 and 359; (f) SEQ ID NOs: 368 and 369; (g) SEQ ID NOs: 378 and 379; and (h) SEQ ID NOs: 388 and 389. The PD-1 antigen-binding protein may comprise a constant region comprising an amino acid sequence of any one of SEQ ID NOs: 265-267, 282, 284-311. The PD-1 antigen-binding protein may comprise a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 320 and 321; (b) SEQ ID NOs: 330 and 331; (c) SEQ ID NOs: 340 and 341; (d) SEQ ID NOs: 350 and 351; (e) SEQ ID NOs: 360 and 361; (f) SEQ ID NOs: 370 and 371; (g) SEQ ID NOs: 380 and 381; and (h) SEQ ID NOs: 390 and 391.

[0266] Also described is a conjugate comprising a PD-1 antigen-binding protein as described in the foregoing paragraphs and a heterologous moiety. Also described is a fusion polypeptide or fusion protein comprising a PD-1 antigen-binding protein as described in the foregoing paragraphs and a heterologous polypeptide or peptide. Also described is a nucleic acid comprising a nucleotide sequence encoding the PD-1 antigen-binding protein, the conjugate, or the fusion polypeptide or fusion protein, as described in the foregoing paragraphs. The nucleic acid may comprise the sequence of any one of SEQ ID NOs: 392-471. Also describes is a vector comprising the nucleic acid, as described above, as well as a host cell comprising the nucleic acid or the vector, as described above. Also described is a kit comprising a PD-1 antigen-binding protein as described in the foregoing paragraphs, or a conjugate, fusion polypeptide, fusion protein, nucleic acid, vector, or host cell, or a combination thereof, and a container. Also described is a pharmaceutical composition that may comprise a PD-1 antigen-binding protein as described in the foregoing paragraphs, or a conjugate, fusion polypeptide, fusion protein, nucleic acid, vector, or host cell, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. Further described is a method of making a PD-1 antigen-binding protein, wherein the method may comprise culturing the host cell as described in this paragraph, so as to express the PD-1 antigen-binding protein and harvesting the expressed PD-1 antigen-binding protein. Additionally described is a method of treating a subject in need thereof, wherein the method may comprise administering to the subject in need thereof a pharmaceutical composition as described in this paragraph in an amount effective to treat the subject. The subject may have a solid tumor and the pharmaceutical composition may be administered to the subject in an amount effective to treat the solid tumor in the subject. Optionally, the the solid tumor is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma.

[0267] The following examples are given merely to illustrate the present disclosure and not in any way to limit its scope.EXAMPLES EXAMPLE 1

[0268] This example demonstrates that combination therapy comprising a PD-1 blocking antibody and recombinant IL-21 is superior to a corresponding monotherapy.

[0269] In a preclinical study, the effects of a combination treatment of a monoclonal PD-1 blocking antibody and recombinant murine IL-21 (rmIL-21) was compared to the effects of monotherapy treatment of either the PD-1 blocking antibody or the rmIL-21.

[0270] On Day 1, CT26 / 3E5 colon carcinoma cells were implanted into BALB / cmice to initiate tumor growth. On Day 12, tumors were measured and the mice were randomized into 4 groups (10 mice per group): Group 1 received an intraperitoneal (IP) injection of 300 µg isotype control antibody...

Claims

1. A conjugate comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 2, wherein "X" represents any amino acid, and wherein said IL-21 mutein amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least 1 amino acid.

2. The conjugate of claim 1, wherein the IL-21 mutein comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 7 amino acids.

3. The conjugate of claim 1, wherein the IL-21 mutein comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5, or 6 amino acids.

4. The conjugate of claim 1, wherein the IL-21 mutein comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 1 or 2 amino acids.

5. The conjugate of any one of the preceding claims, wherein the difference(s) between the amino acid sequence of the IL-21 mutein and the amino acid sequence of SEQ ID NO: 1 occur(s) within (A) amino acids 10-15, inclusive, or amino acids 105-123, inclusive, of SEQ ID NO: 2, optionally, wherein the difference(s) occur(s) at amino acids 11, 14, 15, 109, 110, 112, 113, 116, 119, 120, and / or 123 of SEQ ID NO: 2 or (B) amino acids 5-25, inclusive, or amino acids 65-80, inclusive, of SEQ ID NO: 2, optionally, wherein the difference(s) occur(s) at amino acids 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, and / or 80 of SEQ ID NO: 2.

6. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid sequence with one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1).

7. The conjugate of claim 6, wherein the amino acid substitution occurs at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1.

8. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid substitution: a. at position 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an aliphatic amino acid; b. at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, wherein the substitute amino acid is an acidic amino acid; c. at position 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1, wherein the substitute amino acid is a basic amino acid; d. at position 5, 8, 9, 70, or 76 of SEQ ID NO: 1, wherein the substitute amino acid is an aromatic amino acid; e. at position 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid comprising a side chain amide; f. at position 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is a non-aromatic amino acid comprising a side chain hydroxyl; g. at position 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, wherein the substitute amino acid is an imino acid; h. at position 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, wherein the substitute amino acid is an amino acid comprising a sulfur-containing side chain; or i. or a combination thereof.

9. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid substitution with an amino acid at the position according to TABLE A: Amino Acid position of SEQ ID NO: 1Amino Acid (in single letter code)Amino Acid position of SEQ ID NO: 1Amino Acid (in single letter code)5A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y72D, G, or P8A, D, E, N, S, T, V, or Y73A, D, E, G, H, I, N, P, Q, S, or V9A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y75D, G, or P11D or S76A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V, or Y12A, D, E, N, S, T, or V77D, G, or P13D78D14A, D, or S79D15A, E, I, M, N, Q, S, T, or V80G, or P16D or E109K19D110D23D112D65D, G, or P113K66D, G, or P116A, D, E, I, K, L, M, N, S, T, or V68Q117D69D, G, or P119A, D, E, M, N, Q, S, or T70E, G, P, or Y120D71L123D10. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises: (a) an amino acid sequence of any one of SEQ ID NOs: 3-21, 23-56, 58-112, 114-198, 249-254, and 283; or (b) an amino acid sequence of any one of SEQ ID NOs: 199 to 208, 210 to 222, 224 to 248, and 255.

11. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid sequence with two amino acid substitutions relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), optionally, wherein the amino acid substitution occurs at two of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1.

12. The conjugate of claim 11, wherein the amino acid substitution occurs at two of positions 5, 9, 73, and 76 of SEQ ID NO: 1.

13. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises any two of the following: (i) an amino acid at position 5 of SEQ ID NO: 1, wherein said amino acid is selected from the group consisting of: A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y; (ii) an amino acid at position 9 of SEQ ID NO: 1, wherein said amino acid is selected from the group consisting of: A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y; (iii) an amino acid at position 73 of SEQ ID NO: 1, wherein said amino acid is selected from the group consisting of: A, D, E, G, H, I, N, P, Q, S, or V; and (iv) an amino acid at position 76 of SEQ ID NO: 1, wherein said amino acid is selected from the group consisting of: A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V, or Y.

14. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid substitution at position 5, 9, or 73 of SEQ ID NO: 1, wherein the substitute amino acid is selected from: Amino Acid position of SEQ ID NO: 1Amino Acid (in single letter code)5A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y9A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y73A, D, E, G, H, I, N, P, Q, S, or V15. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid substitution at position 76 of SEQ ID NO: 1 and the substitute amino acid at position 76 is an aliphatic amino acid or an acidic amino acid, optionally, wherein the aliphatic amino acid is alanine or wherein the acidic amino acid is glutamic acid.

16. The conjugate of any one of the preceding claims, wherein the IL-21 mutein is directly attached to the Fc of the antibody.

17. The conjugate of any one of the preceding claims, wherein the IL-21 mutein is attached to the Fc of the antibody via a linker.

18. The conjugate of claim 16 or 17, wherein said conjugate comprises a single IL-21 mutein, wherein said single IL-21 mutein is linked to the C-terminus of one of the two antibody heavy chains.

19. The conjugate of claim 16 or 17, wherein said conjugate comprises two IL-21 muteins, wherein the first IL-21 mutein is linked to the C-terminus of the first antibody heavy chain, and the second IL-21 mutein is linked to the C-terminus of the second antibody heavy chain, optionally, wherein said first IL-21 mutein has the same amino acid sequence as the second IL-21 mutein.

20. The conjugate of claim 19, wherein said first IL-21 mutein and said second IL-21 mutein have the same amino acid sequence.

21. The conjugate of claim 19, wherein said first IL-21 mutein and said second IL-21 mutein have different amino acid sequences.

22. The conjugate of any one of the preceding claims, wherein the IL-21 mutein comprises an amino acid sequence of any one of SEQ ID NOs: 233-245.

23. The conjugate of claim 22, wherein the IL-21 mutein comprises an amino acid sequence of SEQ ID NO: 244.

24. The conjugate of claim 22, wherein the IL-21 mutein comprises an amino acid sequence of SEQ ID NO: 245.

25. The conjugate of any one of the preceding claims, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody.

26. The conjugate of claim 25, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO: 391, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 498-500 or 519; or (ii) two light chains, each comprising the light chain of SEQ ID NO: 391, a heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 501-506, and a heavy chain comprising the heavy chain of any one of SEQ ID NOs: 556-558; or (iii) two light chains, each comprising the light chain of SEQ ID NO: 371, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 508-512; or (iv) two light chains, each comprising the light chain of SEQ ID NO: 371, a heavy chain fused to an IL-21 mutein comprising an amino acid sequence of any one of SEQ ID NOs: 513-518, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 559-561; (v) two light chains, each comprising the light chain variable region of SEQ ID NO: 389, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 498-500 or 519; or (vi) two light chains, each comprising the light chain variable region of SEQ ID NO: 389, a heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 501-506, and a heavy chain comprising the heavy chain of any one of SEQ ID NOs: 556-558; or (vii) two light chains, each comprising the light chain variable region of SEQ ID NO: 369, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 508-512; or (viii) two light chains, each comprising the light chain variable region of SEQ ID NO: 369, a heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 513-518, and a heavy chain comprising the heavy chain of any one of SEQ ID NOs: 559-561; or (ix) two light chains, each comprising a light chain variable region of SEQ ID NO: 389 and a light chain constant region of SEQ ID NO: 391, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 498-500 or 519; or (x) two light chains, each comprising a light chain variable region of SEQ ID NO: 389 and a light chain constant region of SEQ ID NO: 391, a heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 501-506, and a heavy chain comprising the heavy chain of any one of SEQ ID NOs: 556-558; or (xi) two light chains, each comprising a light chain variable region of SEQ ID NO: 369 and a light chain constant region of SEQ ID NO: 371, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 508-512; or (xii) two light chains, each comprising a light chain variable region of SEQ ID NO: 369 and a light chain constant region of SEQ ID NO: 371, a heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of any one of SEQ ID NOs: 513-518, and a heavy chain comprising the heavy chain of any one of SEQ ID NOs: 559-561.

27. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising a LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequence of SEQ ID NOs: 385, 386, and 387, respectively; (ii) two heavy chains, each comprising a HC CDR1, HC CDR2, and HC CDR3 comprising the amino acid sequence of SEQ ID NOs:382, 383, and 384, respectively; and (iii) an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 244, wherein the IL 21 is fused to one of the antibody heavy chains.

28. The conjugate of claim 27, wherein each light chain comprises the light chain variable region of SEQ ID NO: 389 and each heavy chain comprises the heavy chain variable region of SEQ ID NO: 388.

29. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO: 391; (ii) one heavy chain comprising the heavy chain of SEQ ID NO: 556; and (iii) one heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 501.

30. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein: (a) the fusion protein comprises: (i) two light chains, each comprising the light chain variable region of SEQ ID NO: 389; (ii) one heavy chain comprising the heavy chain of SEQ ID NO: 556; and (iii) one heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 501; or (b) the fusion protein comprises: (i) two light chains, each comprising the light chain variable region of SEQ ID NO: 389 and the light chain constant region of SEQ ID NO: 391; (ii) one heavy chain comprising the heavy chain of SEQ ID NO: 556; and (iii) one heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 501.

31. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO: 391, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 499, or (ii) two light chains, each comprising the light chain variable region of SEQ ID NO: 389, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 499, or (iii) two light chains, each comprising the light chain variable region of SEQ ID NO: 389 and the light chain constant region of SEQ ID NO: 391, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 499, or (iv) two light chains, each comprising the light chain of SEQ ID NO: 391, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 519; or (v) two light chains, each comprising the light chain variable region of SEQ ID NO: 389, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 519; or (vi) two light chains, each comprising the light chain variable region of SEQ ID NO: 389 and the light chain constant region of SEQ ID NO: 391, and two heavy chain-IL-21 mutein fusions, each comprising the amino acid sequence of SEQ ID NO: 519.

32. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising a LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequence of SEQ ID NOs: 365, 366, and 367, respectively; (ii) two heavy chains, each comprising a HC CDR1, HC CDR2, and HC CDR3 comprising the amino acid sequence of SEQ ID NOs:362, 363, and 364, respectively; and (iii) an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 244, wherein the IL 21 is fused to one of the antibody heavy chains.

33. The conjugate of claim 32, wherein each light chain comprises the light chain variable region of SEQ ID NO: 369 and each heavy chain comprises the heavy chain variable region of SEQ ID NO: 368.

34. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein: (a) the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO: 371; (ii) one heavy chain comprising the heavy chain of SEQ ID NO: 559; and (iii) one heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 513; or (b) the fusion protein comprises: (i) two light chains, each comprising the light chain variable region of SEQ ID NO: 369; (ii) one heavy chain comprising the heavy chain of SEQ ID NO: 559; and (iii) one heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 513; or (c) the fusion protein comprises: (i) two light chains, each comprising the light chain variable region of SEQ ID NO: 369 and the light chain constant region of SEQ ID NO: 371; (ii) one heavy chain comprising the heavy chain of SEQ ID NO: 559; and (iii) one heavy chain fused to an IL-21 mutein comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 513.

35. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO: 371, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 508, or (ii) two light chains, each comprising the light chain variable region of SEQ ID NO: 369, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 508, or (iii) two light chains, each comprising the light chain variable region of SEQ ID NO: 369 and the light chain constant region of SEQ ID NO: 371, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 508, or (iv) two light chains, each comprising the light chain of SEQ ID NO: 371, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 511; or (v) two light chains, each comprising the light chain variable region of SEQ ID NO: 369, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 511; or (vi) two light chains, each comprising the light chain variable region of SEQ ID NO: 369 and the light chain constant region of SEQ ID NO: 371, and two heavy chain-IL-21 mutein fusions, each comprising the heavy chain fused to an IL-21 mutein of SEQ ID NO: 511.

36. A kit comprising the conjugate of any one of the preceding claims and a container.

37. A pharmaceutical composition comprising the conjugate of any one of the preceding claims and a pharmaceutically acceptable carrier, excipient, or diluent.

38. The pharmaceutical composition of claim 37 for use in treating a subject in need thereof.

39. The pharmaceutical composition of claim 38, for use in treating a subject having a solid tumor.