Anti-PD-1 antibodies and fusion proteins

Abstract

To provide a compound that modulates PD-1 activity.SOLUTION: Provided herein are recombinant antibodies, antigen-binding fragments, and fusion proteins thereof useful for binding to and inhibiting programmed death 1 (PD-1), nucleic acid molecules encoding the same and therapeutic compositions thereof, and methods of using such antibodies, including methods for enhancing a T cell and NK cell function to increase cell and cytokine mediated immunity, and methods of treatment of various immune dysfunction related disorders including cancer and infectious diseases.SELECTED DRAWING: Figure 21A

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 043,114, filed on June 23, 2020, and U.S. Provisional Patent Application No. 63 / 111,459, filed on November 9, 2020, which are hereby incorporated by reference in their entirety.

[0002] The present disclosure generally relates to the fields of molecular biology and medicine. More specifically, the present disclosure provides a fusion protein comprising an anti - PD - 1 antibody or an antigen - binding fragment thereof linked to an IL - 15 polypeptide, wherein the IL - 15 polypeptide is then linked to an interleukin - 15 receptor alpha (IL - 15Rα) polypeptide comprising an IL - 15Rα sushi domain. Further, the present disclosure provides an antibody and an antigen - binding fragment thereof that specifically binds to PD - 1 and fusion proteins comprising such anti - PD - 1 antibodies and their PD - 1 - binding fragments. Also disclosed are antibody fusions or therapeutic compositions comprising an antibody for treating a disease.

Background Art

[0003] PD-1 is a 55KD type I transmembrane protein of 286 amino acids and contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM). The PD-1 cytosolic domain contains two tyrosines, and the most proximal tyrosine (VAYEEL in murine PD-1) is located within the ITIM. The human PD-1 protein and the murine PD-1 protein share approximately 60% amino acid identity, and residues defining four potential N-glycosylation sites and the Ig-V domain are conserved. The ITIM-like motif and the C-terminal tyrosine surrounding the ITIM in the cytoplasmic region are also conserved between the human ortholog and the murine ortholog. PD-1 is a member of the CD-28 family of receptors and is expressed primarily on mature T cells in peripheral tissues and the tumor microenvironment. PD-1 is also expressed on other non-T cell subsets including B cells, specialized antigen-presenting cells (APCs), and natural killer (NK) cells.

[0004] The ligands of PD-1 include the B7 family members PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). PD-L1 is expressed mainly in both lymphoid and non-lymphoid tissues, such as CD4 and CD8 T cells, macrophage lineage cells, peripheral tissues, as well as tumor cells, virus-infected cells and autoimmune tissue cells. However, PD-L2 has a more restricted expression than PD-L1 and is expressed only in macrophages and activated dendritic cells. PD-1 ligands are expressed in a number of human cancers, including melanoma, glioma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, leukemia, pancreatic cancer, renal cell carcinoma and hepatocellular carcinoma, and can be induced in almost all cancers. The interaction between PD-1 and its ligands results in dephosphorylation and inactivation of the T cell kinase ZAP70 and recruitment of SHP2. SHP2 directly dephosphorylates PI3K, inhibits downstream activation of Aktin, causes a decrease in tumor-infiltrating lymphocytes and a decrease in T cell receptor-mediated proliferation, leading to immune evasion. Inhibiting the interaction between PD-1 and PD-L1 reverses immunosuppression, and the effect may be additive when the interaction between PD-1 and PD-L2 is similarly blocked.

[0005] To treat diseases involving PD-1-mediated immunosuppression, several commercially available anti-PD-1 antibodies are currently in use. However, only a subset of patients respond well to these therapies. Depending on the indication and other factors, the typical response rate of patients to each monotherapy ranges from 10% to 30%. One possible reason for this lack of response may be the absence of an inflammatory tumor microenvironment (TME) containing activated anti- tumor CD8+ and CD4+ T cells and other effectors, such as NK cells.

[0006] Combination therapies involving a PD-1 antibody along with various cytokines such as IL-2, IL-15, IL-21, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) may have some efficacy in the treatment of cancer and infectious diseases. However, these therapies are limited by both the high blood concentrations of cytokines required to obtain efficacy and the systemic toxicity associated with the lack of specificity of the administered cytokines for the diseased cells and tissues.

[0007] IL-15 is a 12.5 KD glycoprotein with 114 amino acids and belongs to the cytokine 4α-helix bundle family that also includes IL-2, IL-4, IL-7, IL-9, granulocyte colony-stimulating factor (G-CSF), and GM-CSF. IL-15 is secreted by macrophages, dendritic cells, and monocytes. IL-15 can stimulate central memory CD8 cells to exert immunity without modulating the effects on other T cells. Furthermore, IL-15 can activate NK cells and effector and memory CD8 T cells and rescue T cells from apoptosis induced by regulatory T cells (Tregs). Administration of IL-15 is also associated with a lower risk of inducing systemic toxicity at high doses compared to other cytokines. Human IL-15 can be either soluble or membrane-bound. The membrane-bound form of IL-15, which is the major form, is formed by the direct binding of IL-15 to the cell membrane or by the presentation of IL-15 by the membrane-bound IL-15R receptor.

[0008] The IL-15 receptor is composed of three subunits: IL-15Rα, IL-15Rβ, and IL-15Rγ. IL-15 usually forms a complex with the IL-15 receptor α expressed on APCs before binding to the functional IL-15Rβ and γ subunits on T cells and NK cells. IL-15 can bind to the IL-15Rα receptor alone with an affinity (Ka = 1.1011M -1 )). It can also bind to the IL-15Rβγc signaling complex with a lower affinity (Ka = 1.109M -1) can be combined. The sushi domain (29.5KD) of IL-15Rα plays an important role in the formation of the complex of IL-15 and IL-15Rα.

[0009] One of the limitations when using systemic IL-15 treatment is its extremely short in vivo half-life. Therefore, it is necessary to generate a suitable immunostimulatory form of IL-15 / IL-15Rα with a longer in vivo half-life while retaining its ability to modulate the immune response. Furthermore, an effective IL-15 antagonist that can be selectively targeted to the disease site is needed to avoid unnecessary systemic toxicity and provide a more effective therapeutic benefit.

Summary of the Invention

Problems to be Solved by the Invention

[0010] Compounds that modulate PD-1 activity have the potential as therapeutic agents for the treatment of various diseases and disorders including cancer, inflammation and autoimmune diseases. There is an important, unmet need to develop novel strategies to target various effector molecules to the disease site in order to provide a therapeutic benefit without the side effects associated with non-specific immune activity.

Means for Solving the Problems

[0011] Fusion proteins, antibodies and antigen-binding fragments thereof that bind to PD-1 are provided herein. In embodiments, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. Also disclosed are anti-PD-1 antibodies and antigen-binding fragments thereof that bind to PD-1. Also provided are therapeutic compositions of such fusion proteins, antibodies and antigen-binding fragments thereof, as well as methods of using these fusion proteins, antibodies and antigen-binding fragments thereof.

[0012] In one aspect, the present disclosure provides a fusion protein comprising a PD-1 binding protein, an IL-15 receptor (IL-15R) binding protein (e.g., IL-15 or its IL-15R binding moiety), and an IL-15 binding protein (e.g., IL-15Rα or its IL-15 binding moiety comprising the IL-15Rα sushi domain). In one aspect, the PD-1 binding protein is an antibody or its PD-1 binding fragment. In one embodiment, the IL-15 receptor binding moiety is IL-15. In embodiments, IL-15 is directly or indirectly linked to the sushi domain of IL-15Rα.

[0013] In one embodiment, the fusion protein comprises an anti-PD-1 antibody and an IL-15 polypeptide, and the C-terminus of the IL-15 polypeptide is covalently linked directly or via a polypeptide linker to the N-terminus of one of the heavy chain variable regions of the antibody heavy chain (i.e., the antibody heavy chains form a heterodimer, and one of the heavy chains is linked to the IL-15 polypeptide). In one embodiment, the N-terminus of the IL-15 polypeptide is linked to the C-terminus of the IL-15Rα sushi domain polypeptide.

[0014] In one embodiment, the fusion protein comprises an anti-PD-1 antibody and two IL-15 polypeptides, and each IL-15 polypeptide is covalently linked to the N-terminus of the antibody heavy chain.

[0015] In embodiments where only one heavy chain is linked to the IL-15 / IL-15Rα sushi domain, the Fc domain of the antibody may contain one or more amino acid substitutions that promote heterodimer formation (i.e., association with a heavy chain lacking the fusion of the heavy chain fusion). In embodiments, the amino acid residues in the CH3 domain (CH3-1) of one heavy chain contain amino acid substitutions that replace the amino acid with an amino acid residue having a larger side chain volume, thereby creating a "knob" within the CH3 domain, which is located within a "hole" created by replacing amino acid residues in the CH3 domain (CH3-2) of the other heavy chain with amino acid residues having a smaller side chain volume. In embodiments, the amino acid substitutions are selected from the following group: (1) CH3 domain of the first heavy chain: S354C, T366W; CH3 domain of the second heavy chain: Y349C, T366S, L368A, Y407V (2) CH3 domain of the first heavy chain: T350V, L351Y, F405A, Y407V; CH3 domain of the second heavy chain: T350V, T366L, K392L, T394W (3) CH3 domain of the first heavy chain: L351Y, F405A, Y407V; CH3 domain of the second heavy chain: T366L, K392L, T394W.

[0016] Either the first or the second heavy chain is linked to the IL-15 / IL-15Rα sushi domain.

[0017] In embodiments, the IL-15Rα polypeptide and the IL-15 polypeptide are linked by a first linker. In embodiments, the IL-15 polypeptide and the anti-PD-1 antibody or antigen-binding portion thereof are linked by a second linker. In embodiments, the length of the linker can independently be between 10 and 40 amino acids. In embodiments, the length of the linker is 25 to 35 amino acids. In embodiments, the linker sequence is selected from the group consisting of Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L) and Gln (Q), most preferably, near neutral amino acids selected from the group consisting of Gly (G), Asn (N) and Ser (S). In certain embodiments, the linker sequence is glycine- and serine-rich, and in some embodiments, the linker contains only serine and glycine residues.

[0018] In certain embodiments, the IL-15 and / or IL-15Rα sushi domain contains one or more amino acid substitutions. In embodiments, the amino acid substitutions are located at positions 1, 4, 8, 30, 45, 61, 64, 65 and / or 108 of IL-15. In some embodiments, the amino acid substitutions are located at positions 45, 65 and / or 108 of IL-15. In some embodiments, the amino acid substitutions are one or more of the N1D, N4D, D8N, D30N, D61N, E64Q, L45A, N65S / A / D / K and / or Q108S / E substitutions in IL-15. In some embodiments, the amino acid substitutions are the L45A, N65S / A / D / K and / or Q108S substitutions in IL-15. In one embodiment, the amino acid substitution is the N65S substitution in IL-15.

[0019] In one embodiment, the amino acid substitution is the N60 substitution in the IL-15Rα sushi domain. In one embodiment, the amino acid substitution is the N60A substitution in the IL-15Rα sushi domain.

[0020] In an embodiment, the fusion protein comprises an IL-15 polypeptide comprising one of the following amino acid substitutions: N65S, N65A or N65D. In an embodiment, the fusion protein comprises an IL-15 polypeptide comprising one or more of the following amino acid substitutions: (1) L45A, (2) N65S, N65A or N65D and (3) Q108S. In one embodiment, the fusion protein comprises an IL-15Rα sushi domain polypeptide comprising an N60A amino acid substitution.

[0021] In one aspect, the present disclosure (i) an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, each of the heavy chain and light chain variable regions comprising CDR1, CDR2 and CDR3; (ii) an IL-15 polypeptide or a derivative thereof; (iii) an IL-15Rα sushi domain polypeptide or a derivative thereof; and (iv) a linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide or a derivative thereof and relates to a fusion protein comprising the same.

[0022] In one aspect, the present disclosure (i) an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, each of the heavy chain and light chain variable regions comprising CDR1, CDR2 and CDR3; (ii) an IL-15 polypeptide comprising an amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) an IL-15Rα sushi domain polypeptide comprising an amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; and (iv) a first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide and relates to a fusion protein comprising the same.

[0023] The portion of the anti-PD-1 antibody or its antigen-binding fragment, fusion protein may include the corresponding heavy and light chain variable regions and / or CDRs provided in Figure 1 or otherwise described herein, and may be any antibody or its antigen-binding fragment that specifically binds to PD-1. In embodiments, the fusion protein includes an anti-PD-1 antibody or its antigen-binding fragment, and the CDRs of the heavy and light chain variable regions include the following sequences: The sequence of CDR1 of the heavy chain variable region (CDR1H) includes the sequence of SEQ ID NO: 21; The sequence of CDR2 of the heavy chain variable region (CDR2H) includes the sequence of SEQ ID NO: 22; The sequence of CDR3 of the heavy chain variable region (CDR3H) includes the sequence of SEQ ID NO: 23; The sequence of CDR1 of the light chain variable region (CDR1L) includes the sequence RX 13 X 14 X 15 X 16 IX 17 X 18 WX 19 X 20 (SEQ ID NO: 135); X 13 is A or V; X 14 is S or G; X 15 is Q, E or R; X 16 is G, S, D or N; X 17 is G, S or N; X 18 is S, I, R, T, K, P, N, H or V; X 19 is L or V; X 20 is G or A; The sequence of CDR2 of the light chain variable region (CDR2L) includes the sequence X 21 AX 22 X 23 X 24 X 25 X 26 (SEQ ID NO: 136); X 21is S, D, E or A; X 22 is S or K; X 23 is S, N, T, R or D; X 24 is L or V; X 25 is Q, E or H; X 26 is S, N, A, R, P or T; The sequence of CDR3 (CDR3L) of the light chain variable region is the sequence QQX 27 X 28 SFPX 29 X 30 (SEQ ID NO: 137); X 27 is A or G; X 28 is N, D or Y; X 29 is F or L; X 30 is A or T.

[0024] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO: 96; The sequence of CDR2L comprises the sequence of SEQ ID NO: 97; The sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

[0025] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region comprises the sequence of SEQ ID NO: 24 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; The light chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 98.

[0026] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOs: 21, 22 and 23, respectively; The sequences of CDR1L, CDR2L and CDR3L are SEQ ID NOs: 45, 46 and 47; SEQ ID NOs: 66, 67 and 68; SEQ ID NOs: 70, 67 and 71; SEQ ID NOs: 73, 74 and 75; SEQ ID NOs: 77, 78 and 47; SEQ ID NOs: 80, 81 and 82; SEQ ID NOs: 77, 78 and 84; SEQ ID NOs: 77, 86 and 47; SEQ ID NOs: 88, 89 and 47; SEQ ID NOs: 66, 67 and 47; SEQ ID NOs: 80, 92 and 75; SEQ ID NOs: 80, 94 and 71; SEQ ID NOs: 99, 100 and 47; SEQ ID NOs: 102, 103 and 104; SEQ ID NOs: 106, 103 and 47; SEQ ID NOs: 108, 103 and 47; SEQ ID NOs: 110, 111 and 75; SEQ ID NOs: 77, 103 and 113; SEQ ID NOs: 77, 111 and 47; SEQ ID NOs: 116, 67 and 47; SEQ ID NOs: 118, 119 and 47; SEQ ID NOs: 80, 78 and 47; SEQ ID NOs: 122, 103 and 47; SEQ ID NOs: 124, 125 and 75; SEQ ID NOs: 127, 38 and 68; SEQ ID NOs: 129, 130 and 47 or SEQ ID NOs: 132, 133 and 75, respectively.

[0027] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; The light chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134 or any one of SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134.

[0028] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence RSSX1SLLX2SNGX3X4YLD (SEQ ID NO: 62), where X1 is Q or E; where X2 is H or Y; where X3 is Y or N; where X4 is T or N; The sequence of CDR2L comprises the sequence X5X6SX7X8X9X 10 (SEQ ID NO: 63), where X5 is L, Q or E; where X6 is S, A or V; where X7 is H, N, T or S; where X8 is R or L; where X9 is G, A or H; X 10 is S or T; The sequence of CDR3L comprises the sequence MQGXX 11 X 12 WPYT (SEQ ID NO: 64), X 11is A, T or S; X 12 is H or R.

[0029] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence of SEQ ID NO: 41; The sequence of CDR2L comprises the sequence of SEQ ID NO: 49; The sequence of CDR3 (CDR3L) of the light chain variable region comprises the sequence of SEQ ID NO: 50.

[0030] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region comprises the sequence of SEQ ID NO: 20 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20; The light chain variable region comprises the sequence of SEQ ID NO: 51 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 51.

[0031] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequences of CDR1H, CDR2H and CDR3H each comprise SEQ ID NOs: 17, 18 and 19; The sequences of CDR1L, CDR2L and CDR3L each comprise SEQ ID NOs: 41, 42 and 43; SEQ ID NOs: 41, 52 and 53; SEQ ID NOs: 41, 55 and 56; or SEQ ID NOs: 58, 59 and 60.

[0032] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: the heavy chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20; the light chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 44, 54, 57 or 61.

[0033] In another aspect, the present disclosure provides an anti-PD-1 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof binds to PD-1, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, each of the heavy and light chain variable regions comprises CDR1, CDR2 and CDR3, and the antibody heavy chain comprises a constant region comprising three constant domains CH1, CH2 and CH3.

[0034] In an embodiment, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof comprise the following sequences: the sequence of CDR1H comprises the sequence of SEQ ID NO: 21; the sequence of CDR2H comprises the sequence of SEQ ID NO: 22; the sequence of CDR3H comprises the sequence of SEQ ID NO: 23; the sequence of CDR1L comprises the sequence RX 13 X 14 X 15 X 16 IX 17 X 18 WX 19 X 20 (SEQ ID NO: 135); X 13 is A or V; X 14 is S or G; X 15 is Q, E or R; X 16 is G, S, D or N; X 17 is G, S or N; X 18 is S, I, R, T, K, P, N, H or V; X 19 is L or V; X 20 is G or A; The sequence of CDR2L includes the sequence X 21 AX 22 X 23 X 24 X 25 X 26 (SEQ ID NO: 136); X 21 is S, D, E or A; X 22 is S or K; X 23 is S, N, T, R or D; X 24 is L or V; X 25 is Q, E or H; X 26 is S, N, A, R, P or T; The sequence of CDR3L includes the sequence QQX 27 X 28 SFPX 29 X 30 (SEQ ID NO: 137); X 27 is A or G; X 28 is N, D or Y; X 29 is F or L; X 30 is A or T.

[0035] In an embodiment, the CDRs of the anti-PD-1 antibody or its antigen-binding fragment include the following sequences: The sequence of CDR1H includes the sequence of SEQ ID NO: 21; The sequence of CDR2H includes the sequence of SEQ ID NO: 22; The sequence of CDR3H includes the sequence of SEQ ID NO: 23; The sequence of CDR1L includes the sequence of SEQ ID NO: 96; The sequence of CDR2L includes the sequence of SEQ ID NO: 97; The sequence of CDR3L includes the sequence of SEQ ID NO: 82.

[0036] In an embodiment, the anti-PD-1 antibody or an antigen-binding fragment thereof is as follows: a heavy chain variable region comprising SEQ ID NO: 24 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; and a light chain variable region comprising SEQ ID NO: 98 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 98 is included.

[0037] In an embodiment, the CDRs of the anti-PD-1 antibody or an antigen-binding fragment thereof include the following sequences: The sequences of CDR1H, CDR2H and CDR3H each include SEQ ID NOs: 21, 22 and 23 respectively; The sequences of CDR1L, CDR2L and CDR3L are respectively SEQ ID NOs: 45, 46 and 47; SEQ ID NOs: 66, 67 and 68; SEQ ID NOs: 70, 67 and 71; SEQ ID NOs: 73, 74 and 75; SEQ ID NOs: 77, 78 and 47; SEQ ID NOs: 80, 81 and 82; SEQ ID NOs: 77, 78 and 84; SEQ ID NOs: 77, 86 and 47; SEQ ID NOs: 88, 89 and 47; SEQ ID NOs: 66, 67 and 47; SEQ ID NOs: 80, 92 and 75; SEQ ID NOs: 80, 94 and 71; SEQ ID NOs: 99, 100 and 47; SEQ ID NOs: 102, 103 and 104; SEQ ID NOs: 106, 103 and 47; SEQ ID NOs: 108, 103 and 47; SEQ ID NOs: 110, 111 and 75; SEQ ID NOs: 77, 103 and 113; SEQ ID NOs: 77, 111 and 47; SEQ ID NOs: 116, 67 and 47; SEQ ID NOs: 118, 119 and 47; SEQ ID NOs: 80, 78 and 47; SEQ ID NOs: 122, 103 and 47; SEQ ID NOs: 124, 125 and 75; SEQ ID NOs: 127, 38 and 68; SEQ ID NOs: 129, 130 and 47; or SEQ ID NOs: 132, 133 and 75 are included.

[0038] In an embodiment, the anti-PD-1 antibody or its antigen-binding fragment is as follows: a heavy chain variable region containing SEQ ID NO: 24 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; and a light chain variable region containing SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134 and includes.

[0039] In an embodiment, the CDRs of the anti-PD-1 antibody or its antigen-binding fragment include the following sequences: The sequence of CDR1H includes the sequence of SEQ ID NO: 17; The sequence of CDR2H includes the sequence of SEQ ID NO: 18; The sequence of CDR3H includes the sequence of SEQ ID NO: 19; The sequence of CDR1L includes the sequence RSSX1SLLX2SNGX3X4YLD (SEQ ID NO: 62), and X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L includes the sequence X5X6SX7X8X9X 10 (SEQ ID NO: 63), and X5 is L, Q or E; X6 is S, A or V; X7 is H, N, T or S; X8 is R or L; X9 is G, A or H; X 10 is S or T; The sequence of CDR3L contains the sequence MQGXX 11 X 12 WPYT (SEQ ID NO: 64); X 11 is A, T or S; X 12 is H or R.

[0040] In an embodiment, the CDRs of the anti-PD-1 antibody or its antigen-binding fragment comprise the following sequences: The sequence of CDR1H contains the sequence of SEQ ID NO: 17; The sequence of CDR2H contains the sequence of SEQ ID NO: 18; The sequence of CDR3H contains the sequence of SEQ ID NO: 19; The sequence of CDR1L contains the sequence of SEQ ID NO: 41; The sequence of CDR2L contains the sequence of SEQ ID NO: 49; The sequence of CDR3L contains the sequence of SEQ ID NO: 50.

[0041] In an embodiment, the anti-PD-1 antibody or its antigen-binding fragment is as follows: A heavy chain variable region containing SEQ ID NO: 20 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20; and A light chain variable region containing SEQ ID NO: 51 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 51 is included.

[0042] In an embodiment, the CDRs of the anti-PD-1 antibody or its antigen-binding fragment comprise the following sequences: The sequences of CDR1H, CDR2H and CDR3H each contain SEQ ID NOs: 17, 18 and 19 respectively; The sequences of CDR1L, CDR2L, and CDR3L include SEQ ID NO: 41, 42, and 43; SEQ ID NO: 41, 52, and 53; SEQ ID NO: 41, 55, and 56; or SEQ ID NO: 58, 59, and 60, respectively.

[0043] In an embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is as follows: a heavy chain variable region comprising SEQ ID NO: 20 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20; and a light chain variable region comprising SEQ ID NO: 44, 54, 57, or 61 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 44, 54, 57, or 61 comprising.

[0044] In one aspect, the present disclosure is (i) an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2, and CDR3; wherein: the sequence of CDR1H comprises the sequence of SEQ ID NO: 21; the sequence of CDR2H comprises the sequence of SEQ ID NO: 22; the sequence of CDR3H comprises the sequence of SEQ ID NO: 23; the sequence of CDR1L comprises the sequence of SEQ ID NO: 96; the sequence of CDR2L comprises the sequence of SEQ ID NO: 97; the sequence of CDR3L comprises the sequence of SEQ ID NO: 82 anti-PD-1 antibody or antigen-binding fragment thereof; (ii) an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relating to a fusion protein comprising Optionally, the anti-PD-1 antibody or antigen-binding fragment thereof comprises two heavy chains: (a) the CH3 domain of the first heavy chain comprises the amino acid substitutions S354C and T366W (Kabat EU index numbering); (b) the CH3 domain of the second heavy chain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

[0045] In one aspect, the disclosure provides (i) an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: the sequence of CDR1H comprises the sequence of SEQ ID NO: 21; the sequence of CDR2H comprises the sequence of SEQ ID NO: 22; the sequence of CDR3H comprises the sequence of SEQ ID NO: 23; the sequence of CDR1L comprises the sequence of SEQ ID NO: 96; the sequence of CDR2L comprises the sequence of SEQ ID NO: 97; the sequence of CDR3L comprises the sequence of SEQ ID NO: 82 the anti-PD-1 antibody or antigen-binding fragment thereof; (ii) an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; (iv) A first linker polypeptide that connects an IL-15Rα sushi domain polypeptide and an IL-15 polypeptide; relating to a fusion protein comprising, optionally, the anti-PD-1 antibody or antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain comprises the amino acid substitutions T350V, L351Y, F405A and Y407V (Kabat EU index numbering); (b) The CH3 domain of the second heavy chain comprises the amino acid substitutions T350V, T366L, K392L and T394W (Kabat EU index numbering), comprising.

[0046] In one aspect, the present disclosure provides, (i) An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO: 96; The sequence of CDR2L comprises the sequence of SEQ ID NO: 97; The sequence of CDR3L comprises the sequence of SEQ ID NO: 82 an anti-PD-1 antibody or antigen-binding fragment thereof; (ii) An IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; (iv) A first linker polypeptide that connects an IL-15Rα sushi domain polypeptide and an IL-15 polypeptide; relating to a fusion protein comprising, Optionally, the anti-PD-1 antibody or antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain comprises the amino acid substitutions L351Y, F405A, and Y407V (Kabat EU index numbering); (b) The CH3 domain of the second heavy chain comprises the amino acid substitutions T366L, K392L, and T394W (Kabat EU index numbering).

[0047] In one aspect, the disclosure provides (i) An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2, and CDR3; wherein: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO: 96; The sequence of CDR2L comprises the sequence of SEQ ID NO: 97; The sequence of CDR3L comprises the sequence of SEQ ID NO: 82 An anti-PD-1 antibody or antigen-binding fragment thereof; (ii) An IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, optionally wherein the amino acid N65 of SEQ ID NO: 212 is substituted with S, D, or A; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, optionally wherein the amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) A first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; to a fusion protein comprising Optionally, the anti-PD-1 antibody or antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain contains the amino acid substitutions S354C and T366W (Kabat EU indexing); (b) The CH3 domain of the second heavy chain contains the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU indexing).

[0048] In one aspect, the disclosure relates to (i) an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO: 96; The sequence of CDR2L comprises the sequence of SEQ ID NO: 97; The sequence of CDR3L comprises the sequence of SEQ ID NO: 82 an anti-PD-1 antibody or antigen-binding fragment thereof; (ii) an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, optionally wherein the amino acid N65 of SEQ ID NO: 212 is substituted with S, D or A; (iii) an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, optionally wherein the amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising Optionally, the anti-PD-1 antibody or antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain contains the amino acid substitutions T350V, L351Y, F405A and Y407V (Kabat EU indexing); (b) The CH3 domain of the second heavy chain contains the amino acid substitutions T350V, T366L, K392L, and T394W (Kabat EU indexing).

[0049] In one aspect, the present disclosure relates to (i) an anti-PD-1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2, and CDR3; wherein: the sequence of CDR1H comprises the sequence of SEQ ID NO: 21; the sequence of CDR2H comprises the sequence of SEQ ID NO: 22; the sequence of CDR3H comprises the sequence of SEQ ID NO: 23; the sequence of CDR1L comprises the sequence of SEQ ID NO: 96; the sequence of CDR2L comprises the sequence of SEQ ID NO: 97; the sequence of CDR3L comprises the sequence of SEQ ID NO: 82 the anti-PD-1 antibody or an antigen-binding fragment thereof; (ii) an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, optionally wherein the amino acid N65 of SEQ ID NO: 212 is substituted with S, D, or A; (iii) an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, optionally wherein the amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising Optionally, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain contains the amino acid substitutions L351Y, F405A, and Y407V (Kabat EU indexing). (b) The CH3 domain of the second heavy chain contains the amino acid substitutions T366L, K392L and T394W (Kabat EU indexing).

[0050] In one aspect, the present disclosure is (i) an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; where: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence of SEQ ID NO: 41; The sequence of CDR2L comprises the sequence of SEQ ID NO: 49; The sequence of CDR3L comprises the sequence of SEQ ID NO: 50 an anti-PD-1 antibody or antigen-binding fragment thereof; (ii) an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising Optionally, the anti-PD-1 antibody or antigen-binding fragment thereof is two heavy chains: (a) The CH3 domain of the first heavy chain contains the amino acid substitutions S354C and T366W (Kabat EU indexing); (b) The CH3 domain of the second heavy chain contains the amino acid substitutions Y349C, T366S, L3 68A and Y407V (Kabat EU indexing).

[0051] In one aspect, the present disclosure is (i) an anti-PD-1 antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region and a light-chain variable region, wherein each of the heavy-chain and light-chain variable regions comprises CDR1, CDR2, and CDR3; wherein: the sequence of CDR1H comprises the sequence of SEQ ID NO: 17; the sequence of CDR2H comprises the sequence of SEQ ID NO: 18; the sequence of CDR3H comprises the sequence of SEQ ID NO: 19; the sequence of CDR1L comprises the sequence of SEQ ID NO: 41; the sequence of CDR2L comprises the sequence of SEQ ID NO: 49; the sequence of CDR3L comprises the sequence of SEQ ID NO: 50 the anti-PD-1 antibody or an antigen-binding fragment thereof; (ii) an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising optionally, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises two heavy chains: (a) the CH3 domain of the first heavy chain comprises the amino acid substitutions T350V, L351Y, F405A, and Y407V (Kabat EU index numbering); (b) the CH3 domain of the second heavy chain comprises the amino acid substitutions T350V, T366L, K392L, and T394W (Kabat EU index numbering).

[0052] In one aspect, the present disclosure is as follows: (i) An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence of SEQ ID NO: 41; The sequence of CDR2L comprises the sequence of SEQ ID NO: 49; The sequence of CDR3L comprises the sequence of SEQ ID NO: 50; An anti-PD-1 antibody or antigen-binding fragment thereof; (ii) An IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 214; (iv) A first linker polypeptide that connects the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relating to a fusion protein comprising Optionally, the anti-PD-1 antibody or antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain comprises the amino acid substitutions L351Y, F405A and Y407V (Kabat EU index numbering); (b) The CH3 domain of the second heavy chain comprises the amino acid substitutions T366L, K392L and T394W (Kabat EU index numbering).

[0053] In one aspect, the present disclosure provides (i) An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: The sequence of CDR1H includes the sequence of SEQ ID NO: 17; The sequence of CDR2H includes the sequence of SEQ ID NO: 18; The sequence of CDR3H includes the sequence of SEQ ID NO: 19; The sequence of CDR1L includes the sequence of SEQ ID NO: 41; The sequence of CDR2L includes the sequence of SEQ ID NO: 49; The sequence of CDR3L includes the sequence of SEQ ID NO: 50; An anti-PD-1 antibody or an antigen-binding fragment thereof; (ii) An IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, optionally wherein the amino acid N65 of SEQ ID NO: 212 is substituted with S, D or A; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, optionally wherein the amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) A first linker polypeptide that connects the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising; Optionally, the anti-PD-1 antibody or an antigen-binding fragment thereof has two heavy chains: (a) The CH3 domain of the first heavy chain comprises the amino acid substitutions S354C and T366W (Kabat EU indexing); (b) The CH3 domain of the second heavy chain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU indexing).

[0054] In one aspect, the present disclosure provides (i) An anti-PD-1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: The sequence of CDR1H includes the sequence of SEQ ID NO: 17; The sequence of CDR2H includes the sequence of SEQ ID NO: 18; The sequence of CDR3H includes the sequence of SEQ ID NO: 19; The sequence of CDR1L includes the sequence of SEQ ID NO: 41; The sequence of CDR2L includes the sequence of SEQ ID NO: 49; The sequence of CDR3L includes the sequence of SEQ ID NO: 50 An anti-PD-1 antibody or an antigen-binding fragment thereof; (ii) An IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, wherein optionally the amino acid N65 of SEQ ID NO: 212 is substituted with S, D or A; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, wherein optionally the amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) A first linker polypeptide that connects the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising Optionally, the anti-PD-1 antibody or an antigen-binding fragment thereof has two heavy chains: (a) The CH3 domain of the first heavy chain includes the amino acid substitutions T350V, L351Y, F405A and Y407V (Kabat EU index numbering); (b) The CH3 domain of the second heavy chain includes the amino acid substitutions T350V, T366L, K39 2L and T394W (Kabat EU index numbering).

[0055] In one aspect, the present disclosure provides (i) An anti-PD-1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and light chain variable regions comprises CDR1, CDR2 and CDR3; wherein: The sequence of CDR1H includes the sequence of SEQ ID NO: 17; The sequence of CDR2H includes the sequence of SEQ ID NO: 18; The sequence of CDR3H includes the sequence of SEQ ID NO: 19; The sequence of CDR1L includes the sequence of SEQ ID NO: 41; The sequence of CDR2L includes the sequence of SEQ ID NO: 49; The sequence of CDR3L includes the sequence of SEQ ID NO: 50 An anti-PD-1 antibody or an antigen-binding fragment thereof; (ii) An IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, wherein optionally the amino acid N65 of SEQ ID NO: 212 is substituted with S, D or A; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, wherein optionally the amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) A first linker polypeptide that links the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; relates to a fusion protein comprising Optionally, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises two heavy chains: (a) The CH3 domain of the first heavy chain comprises the amino acid substitutions L351Y, F405A and Y407V (Kabat EU index numbering); (b) The CH3 domain of the second heavy chain comprises the amino acid substitutions T366L, K392L and T394W (Kabat EU index numbering).

[0056] In one embodiment, the anti-PD-1 antibody or an antigen-binding fragment thereof provided by the present disclosure is a multispecific or bispecific antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment is a bispecific antibody comprising a complementary region that binds to PD-L1 or PD-L2. The present disclosure also provides conjugates or fusion molecules of antibodies to, for example, but not limited to, fluorescent labels, contrast agents, therapeutic agents or cytotoxic agents.

[0057] The present disclosure further provides a pharmaceutical composition comprising one or more of an anti-PD-1 antibody or an antigen-binding fragment thereof or a fusion protein comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0058] In embodiments, the present disclosure provides a nucleic acid molecule encoding an anti-PD-1 antibody or an antigen-binding fragment thereof disclosed herein and a nucleic acid molecule encoding a fusion protein disclosed herein, and a vector comprising such a nucleic acid molecule. Also provided are cells comprising a vector encoding an anti-PD-1 antibody or an antigen-binding fragment thereof or a fusion protein disclosed herein.

[0059] In embodiments, the present disclosure provides a method of inhibiting the binding of PD-1 to its ligand, the method comprising administering to a subject in need thereof an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof or a fusion protein disclosed herein.

[0060] In embodiments, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-PD-1 antibody or an antigen-binding fragment thereof or a fusion protein in a pharmaceutically acceptable form.

[0061] In embodiments, the present disclosure provides a method of increasing T cell activation in a subject in need thereof, the method comprising administering an effective amount of an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein. In one embodiment, the present disclosure provides a method of stimulating the immune system in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein in a pharmaceutically acceptable form. In one embodiment, the present disclosure provides a method of reducing viral replication in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein.

[0062] The present disclosure also provides a method of inhibiting the interaction of PD-1 with PD-L1 and / or PD-L2 in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein. The present disclosure further provides a method of inhibiting PD-1-mediated immunosuppression in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein.

[0063] The present disclosure further provides a method of stimulating an immune response against cells or tissues expressing PD-1 in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein. In certain embodiments, the cells or tissues expressing PD-1 are cancerous cells or, without limitation thereto, cells infected with a pathogen, including a virus or bacteria.

[0064] In one aspect, the present disclosure provides a method of treating a patient in need thereof for cancer, immune disorder or infection, the method comprising: (a) treating cells in vitro with an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein; and (b) administering the treated cells to the patient. In some embodiments, the cells are T cells.

[0065] In another aspect, the present disclosure provides a method of enhancing T cell activation in response to an antigen in a subject, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein or a pharmaceutical composition disclosed herein.

[0066] In another aspect, the present disclosure provides a method of treating a condition in a subject who would benefit from upregulation of the immune response of the subject, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein. In certain embodiments, the subject has upregulated expression of PD-L1 or the subject has been identified as positive for expression of PD-L1.

[0067] In one embodiment, the present disclosure provides a method of using an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein to treat a subject who is non-responsive (primary resistance) to therapy with a checkpoint inhibitor and / or a subject who initially responds to checkpoint inhibitor treatment but later becomes resistant (secondary or acquired resistance) to checkpoint inhibitor blockade. The method for such treatment comprises administering to the subject an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein. In some embodiments, the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0068] In one embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein to a subject in need thereof, further comprising administering an additional therapeutic agent or therapy to the subject, wherein the additional therapeutic agent or therapy is selected from the group consisting of cancer vaccines, checkpoint inhibitors, antibodies against tumor-specific antigens, Bacillus Calmette-Guerin (BCG) vaccine, cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory agents, and dietary supplements. In an embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein to a subject in need thereof, further comprising administering to the subject a checkpoint inhibitor selected from CTLA-4, PD-1, PD-L1, and PD-L2 inhibitors. In an embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein to a subject in need thereof, further comprising administering to the subject one or more inhibitors of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, and / or CD160. In an embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein to a subject in need thereof, further comprising administering an additional therapeutic agent or therapy to the subject, wherein the additional therapeutic agent or therapy is administered concurrently or sequentially with the anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein. In an embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein to a subject in need thereof, further comprising administering an additional therapeutic agent or therapy to the subject, wherein the additional therapeutic agent or therapy is administered separately or as a mixture with the anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein.In one embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody or an antigen-binding fragment or fusion protein thereof disclosed herein in combination with chemotherapy, radiation therapy, or surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0069]

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Mode for Carrying Out the Invention

[0070] The present disclosure provides a fusion protein comprising an anti-PD-1 antibody or an antigen-binding fragment thereof linked to an IL-15 polypeptide, which IL-15 polypeptide is then linked to an interleukin-15 receptor alpha (IL-15Rα) polypeptide comprising the IL-15Rα sushi domain. Further, antibodies that specifically bind to PD-1 and antigen-binding fragments thereof, and fusion proteins comprising such anti-PD-1 antibodies and their PD-1 binding fragments, are provided in this specification. are provided in this document.

[0071] Fusion protein comprising an anti-PD-1 antibody or antigen fragment thereof In one aspect, the present disclosure provides a fusion protein comprising an antibody that binds to PD-1 or an antigen-binding fragment thereof. The portion of this fusion protein can be any antibody or antibody fragment that specifically binds to PD-1, including those comprising the corresponding heavy and light chain variable regions or CDRs provided in Figure 1 or otherwise described herein.

[0072] Fusion proteins containing stimulatory domains are disclosed herein. As used herein, a "stimulatory domain" is a domain that promotes an immune response. A stimulatory domain can stimulate an immune response mediated, for example, by inducing T cell or NK cell activity and / or proliferation. In embodiments, the stimulatory domain stimulates cells that respond to interleukins or interferons such as, but not limited to, IL-2, IL-7, IL-15, and IL-21. In one embodiment, the stimulatory domain binds to and stimulates a receptor that is responsive to interleukins or interferons such as, but not limited to, IL-2, IL-7, IL-13, IL-15, and IL-21. The stimulatory domain can also be a hybrid domain that is a heterocomplex of two or more ligands linked to each other by covalent bonds.

[0073] In embodiments, the stimulatory domain comprises a sequence or domain that promotes IL-15 stimulation of the IL-15 receptor (IL-15R).

[0074] In one embodiment, the stimulatory domain that promotes IL-15R stimulation comprises IL-15 or an IL-15 derivative.

[0075] In one embodiment, the stimulatory domain that promotes IL-15R stimulation comprises an IL-15Rα polypeptide comprising the IL-15Rα sushi domain or a derivative thereof. In one embodiment, the stimulatory domain comprises the sushi domain of the IL-15Rα chain.

[0076] In one embodiment, the stimulatory domain comprises IL-15 or a derivative thereof, and the binding is enhanced by the presence of an IL-15Rα polypeptide comprising an IL-15Rα sushi domain or a derivative thereof. In one embodiment, the stimulatory domain comprises a complex of IL-15 or a derivative thereof and an IL-15Rα polypeptide comprising an IL-15Rα sushi domain or a derivative thereof. In some embodiments, the stimulatory domain comprises IL-15 or a derivative thereof and an IL-15Rα polypeptide comprising an IL-15Rα sushi domain or a derivative thereof, and the two polypeptides are covalently linked by a linker (including but not limited to a linker comprising SEQ ID NO: 215).

[0077] In one embodiment, IL-15 or a derivative thereof is located at the N-terminus of an IL-15Rα polypeptide comprising an IL-15Rα sushi domain or a derivative thereof. In one embodiment, IL-15 or a derivative thereof is located at the C-terminus of an IL-15Rα polypeptide comprising an IL-15Rα sushi domain or a derivative thereof. In one embodiment, the stimulatory domain comprises the sequence of the SD15 domain highlighted by any one of SEQ ID NOs: 177-180.

[0078] As demonstrated herein, a fusion protein comprising (1) a PD-1 binding domain that blocks the binding of PD-1 to PD-L1 and inhibits immunosuppression and (2) a stimulatory domain that promotes an immune response is provided herein, and the fusion protein provides an increase in immune cell activity as compared to two separate molecules that provide the aforementioned functions separately. Specifically, the experiments disclosed herein demonstrate that a fusion protein containing a PD-1 binding domain that blocks the binding of PD-1 to PD-L1 and both a stimulatory domain IL-15 or a derivative thereof and an IL-15Rα polypeptide comprising an IL-15Rα sushi domain or a derivative thereof promoted increased proliferation, Th1 cytokine release, and molecules related to the killing activity of NK and T cells as compared to providing the domains in separate molecules.

[0079] In certain embodiments, the fusion protein comprises a stimulatory domain disclosed herein covalently linked via a flexible linker to an anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, provided herein are fusion proteins in which the stimulatory domain disclosed herein is directly fused to an anti-PD-1 antibody or antigen-binding fragment disclosed herein.

[0080] As used herein, "covalently linked" or "fused" refers to the association of two or more polypeptides by a covalent bond. In some embodiments, the two polypeptides linked by a covalent bond are fused directly to each other, i.e., without any additional polypeptide sequences between the first and second peptides. Thus, in some embodiments, the N-terminus of the first polypeptide is directly fused to the C-terminus of the second polypeptide, or vice versa. In other embodiments, the two polypeptides linked by a covalent bond are part of a continuous polypeptide chain but are not fused directly to each other (i.e., the two polypeptides may be separated by one or more amino acids, a linker, or another polypeptide). The term "covalently linked" does not imply a particular orientation of the two or more polypeptides fused to each other.

[0081] IL-15 is a cytokine of 14-15 KD and has structural similarity to IL-2. IL-15 is also known as MGC9721. Various cell types constitutively produce IL-15 mRNA, including monocytes, macrophages, DCs, keratinocytes, epithelial skin cells, fibroblasts, various epithelial cells, bone marrow stromal cells, and nerve cells. Furthermore, IL-15 mRNA is also produced in kidney, placenta, lung, heart, skeletal muscle, and brain tissues. However, only monocytes, DCs, epithelial cells, bone marrow stromal cells, fibroblasts, and very few other cells and tissues secrete detectable levels of IL-15. IL-15 and IL-2 are known to bind to the same hematopoietin subunit and share numerous biological activities. IL-15 regulates T and NK cell activation and proliferation, and the number of CD8 + memory cells is affected by the balance between IL-15 and IL-2. In embodiments, the IL-15 or IL-15 derivative disclosed herein has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the activity of human IL-15. IL-15 can be mammalian IL-15, preferably primate IL-15, and more preferably human IL-15. The amino acid sequence of human IL-15 (accession number NP_000576) is provided as SEQ ID NO: 212.

[0082] The term "IL-15 derivative" refers to a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 212, the mature form of human IL-15. Techniques for making such derivatives are known in the art. In some embodiments, the IL-15 or IL-15 derivative sequence may include one or more amino acid substitutions. In some embodiments, the amino acid substitutions are at positions 1, 4 of IL-15 、8, 30, 45, 61, 64, 65 and / or 108 (residue numbers refer to the mature form of the IL-15 protein). In some embodiments, the amino acid substitution is located at positions 45, 65 and / or 108 of IL-15. In some embodiments, the amino acid substitution in IL-15 is N1D, N4D, D8N, D30N, D61N, E64Q, L45A, N65S / A / D / K and / or Q108S / E. In some embodiments, the amino acid substitution in IL-15 is L45A, N65S / A / D / K and / or Q108S. In embodiments, the fusion proteins disclosed herein comprise an IL-15 polypeptide comprising one of the following amino acid substitutions: N65S, N65A or N65D. In embodiments, the fusion proteins disclosed herein comprise an IL-15 polypeptide comprising one or more of the following amino acid substitutions: L45A, N65S, N65A or N65D and Q108S.

[0083] The amino acid sequence of human IL-15Rα isoform 1 precursor (accession number NP_002180) is provided in SEQ ID NO: 213. The IL-15 receptor, i.e., the IL-15 receptor complex, specifically binds to IL-15 with high affinity and consists of a unique interleukin 15 receptor α subunit, IL-2 / IL-15Rβ and a common γ chain / IL-2Rγ subunit. IL-15Rα is expressed by mitogen-activated macrophages, NK cells, and CD4 + and CD8 + T cells. Human IL-15Rα consists of seven exons, and alternative mRNA splicing can result in eight molecular IL-15Rα isoforms with different extracellular or intracellular domains. The full-length isoform consists of an extracellular portion containing a conserved protein-binding motif (sushi domain), a transmembrane domain, and an intracellular tail.

[0084] As used herein, the term "sushi domain" of IL-15Rα refers to a domain that starts at the first cysteine residue (C1) after the signal peptide of IL-15Rα and ends at the fourth cysteine residue (C4) after the signal peptide. The sushi domain corresponding to a portion of the extracellular region of IL-15Rα is involved in binding to IL-15. The sushi domain in the present disclosure has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the activity of the sushi domain of the human IL-15Rα chain.

[0085] The amino acid sequence of the human IL-15Rα isoform 1 precursor is provided in SEQ ID NO: 213 (this sequence includes the signal sequence). The amino acid sequence of the sushi domain of human IL-15Rα is provided in SEQ ID NO: 214.

[0086] The terms "IL-15Rα sushi domain derivative" or "IL-15Rα sushi domain variant" refer to polypeptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence of the human IL-15Rα sushi domain (SEQ ID NO: 214). Techniques for making such derivatives or variants are known in the art. All such derivatives contain the four cysteine residues of the sushi domain of IL-15Rα. In some such derivatives, naturally occurring amino acids may be replaced by chemically modified amino acids in order to alter the polypeptide half-life. In some embodiments, the IL-15Rα sushi domain or IL-15Rα sushi domain derivative sequence may contain one or more amino acid substitutions. In some embodiments, the amino acid substitution is located at position 60 of IL-15Rα (numbering of IL-15Rα residues includes the signal peptide, see, for example, SEQ ID NO: 213). In some embodiments, the amino acid substitution in the IL-15Rα sushi domain is N60A (i.e., asparagine (N) at amino acid 60 is , replaced with alanine (A)).

[0087] IL-15 binds with high affinity to IL-15Rα, which then associates with the IL-15Rβ / γc complex expressed by the same target cell (cis-presentation). IL-15Rα is also known to trans-present IL-15 with high affinity to different target cells expressing the IL-15Rβ / γ(c) complex (trans-presentation). The IL-15 cis- and trans-presentation mechanisms lead to different kinetics of receptor activation and signaling, with cis-presentation inducing a rapid transient response and trans-presentation inducing a slower, more sustained one.

[0088] In some embodiments, the fusion protein disclosed herein comprises an activating domain comprising a hybrid domain comprising the sushi domain of the IL-15Rα chain attached to IL-15 by a linker (including, but not limited to, the linker of SEQ ID NO: 215). In certain embodiments, the activating domain disclosed herein is covalently linked to an anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein via a second linker (including, but not limited to, the linker of SEQ ID NO: 216). In other embodiments, the activating domain exemplified herein is directly fused to an anti-PD-1 antibody or antigen-binding fragment disclosed herein.

[0089] In one embodiment, provided is an "N-terminal fusion protein" in which the activating domain is linked to the N-terminus of the anti-PD1 binding portion of the fusion protein (e.g., the N-terminus of the heavy chain of an anti-PD-1 antibody disclosed herein) directly or via a linker (e.g., via one or more peptides).

[0090] The N-terminal fusion proteins include, but are not limited to, the following (exemplary) fusion proteins (listed from N- to C-terminus; "-" indicates direct covalent linkage or linkage via a linker (e.g., via one or more peptides)): (1) (IL-15 or a derivative thereof) - (heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) (2) (IL-15 or a derivative thereof) - (light chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) (3) (IL-15Rα polypeptide comprising the IL-15Rα sushi domain or a derivative thereof) - (IL-15 or a derivative thereof) - (heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) (4) (IL-15Rα polypeptide comprising the IL-15Rα sushi domain or a derivative thereof) - (IL-15 or a derivative thereof) - (light chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) (5) (IL-15Rα sushi domain or a derivative thereof) - (IL-15 or a derivative thereof) - (heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) (5) (IL-15 or its derivative) - (IL-15Rα polypeptide containing IL-15Rα sushi domain or its derivative) - (heavy chain of anti-PD-1 antibody or its antigen-binding fragment) (6) (IL-15 or its derivative) - (IL-15Rα polypeptide containing IL-15Rα sushi domain or its derivative) - (light chain of anti-PD-1 antibody or its antigen-binding fragment)

[0091] The N-terminal fusion protein may contain more than one stimulatory domain.

[0092] In one embodiment, there is provided a fusion protein in which a stimulatory domain is covalently linked to the N-terminus of one (and only one) of the heavy chain of an anti-PD-1 antibody or its antigen-binding fragment. An N-terminal fusion protein containing a single stimulatory domain is referred to as a "1-N-terminal fusion protein".

[0093] (1) A stimulatory domain (2) The first heavy chain of an anti-PD-1 antibody or its antigen-binding fragment, wherein the stimulatory domain is covalently linked to the N-terminus of the first heavy chain, and (3) The second heavy chain of an anti-PD-1 antibody or its antigen-binding fragment, wherein the second heavy chain is not linked to the stimulatory domain. In some embodiments, the stimulatory domain comprises (i) IL-15 or its derivative, or (ii) an IL-15Rα polypeptide containing an IL-15Rα sushi domain or its derivative, or (iii) both.

[0094] In one embodiment, there is provided a fusion protein in which a stimulatory domain is covalently linked to the N-termini of both the heavy chain of an anti-PD-1 antibody or its antigen-binding fragment. An N-terminal fusion protein containing two stimulatory domains, both linked to different antibody chains, is referred to as a "2-N-terminal fusion protein".

[0095] In one embodiment, a "C-terminal fusion protein" is provided in which the stimulatory domain is linked to the C-terminus of the anti-PD1 binding portion of the fusion protein (e.g., the C-terminus of the heavy chain of an anti-PD-1 antibody as disclosed herein) directly or via a linker (e.g., via one or more peptides).

[0096] The C-terminal fusion proteins include, but are not limited to, the following (exemplary) fusion proteins (components listed from N- to C-terminus, and "-" indicates a direct covalent linkage or a linkage via a linker (e.g., via one or more peptides): (1) (heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) - (IL-15 or a derivative thereof) (2) (light chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) - (IL-15 or a derivative thereof) (3) (heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) - (IL-15Rα sushi domain or an IL-15Rα polypeptide containing a derivative thereof) - (IL-15 or a derivative thereof) (4) (light chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) - (IL-15Rα sushi domain or an IL-15Rα polypeptide containing a derivative thereof) - (IL-15 or a derivative thereof) (5) (heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) - (IL-15 or a derivative thereof) - (IL-15Rα sushi domain or an IL-15Rα polypeptide containing a derivative thereof) (6) (light chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) - (IL-15 or a derivative thereof) - (IL-15Rα sushi domain or an IL-15Rα polypeptide containing a derivative thereof)

[0097] The C-terminal fusion protein can include more than one stimulatory domain.

[0098] In one embodiment, a fusion protein is provided in which the stimulatory domain is covalently linked to the C-terminus of one (and only one) of the heavy chains of the anti-PD-1 antibody or an antigen-binding fragment thereof. A C-terminal fusion protein containing a single stimulatory domain is referred to as a "1-C-terminal fusion protein".

[0099] (1) A stimulatory domain, (2) the first heavy chain of the anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the stimulatory domain is covalently linked to the C-terminus of the first heavy chain, and (3) the second heavy chain of the anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the second heavy chain is not linked to the stimulatory domain, are provided herein. In some embodiments, the sti mulatory domain comprises (i) IL-15 or a derivative thereof, or (ii) an IL-15Rα sushi domain or a derivative thereof-containing IL-15Rα polypeptide, or (iii) both.

[0100] In one embodiment, a fusion protein is provided in which the stimulatory domain is covalently linked to the C-terminus of both the heavy chains of the anti-PD-1 antibody or an antigen-binding fragment thereof. A C-terminal fusion protein containing two stimulatory domains, both linked to different antibody chains, is referred to as a "2-C-terminal fusion protein".

[0101] Figure 21 depicts a schematic diagram of an exemplary fusion protein as described herein.

[0102] Therapeutic levels of IL-2 are associated with high levels of toxicity upon binding to the IL-2 receptor. Similarly, using therapeutic levels of IL-15 may also cause toxicity because IL-15 shares two receptor subunits with IL-2. However, when presented in the cis position, the N-terminal IL-15 fusion protein can selectively bind to tumor-infiltrating lymphocytes (TILs) while showing lower binding to peripheral T cells, so the N-terminal fusion protein demonstrates reduced toxicity compared to the C-terminal fusion protein. When presented in the cis position, the N-terminal fusion protein can bind to IL-15Rβγ and PD-1 simultaneously while reducing IL-15Rβ / γ binding, enabling the N-terminal fusion protein to selectively bind to TILs and promote TIL proliferation. In contrast, the C-terminal fusion protein may bind equally to both peripheral T cells and TILs and may exhibit higher toxicity than the N-terminal fusion protein (Figure 25). 1 The N-terminal fusion protein shows reduced toxicity compared to the N-terminal fusion protein containing two stimulatory domains covalently linked to the anti-PD-1 antibody disclosed herein (2N-terminal fusion protein). The mutated N-terminal fusion protein shows further reduced toxicity due to even lower binding to peripheral T cells.

[0103] In one aspect, the disclosure provides the following: (i) An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, each of the heavy chain and light chain variable regions comprising CDR1, CDR2, and CDR3, and the antibody heavy chain comprising a constant region comprising three constant domains CH1, CH2, and CH3; (ii) An interleukin 15 (IL-15) polypeptide comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; (iii) An IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 214; and (iv) A first linker polypeptide that connects the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide relates to a fusion protein comprising the same.

[0104] The anti-PD-1 antibody or antigen-binding fragment thereof, the portion of the fusion protein, can be any that specifically binds to PD-1, including those provided in Figure 1 or described herein that contain the corresponding variable regions or CDRs. In embodiments, the fusion protein comprises an antibody or antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence RX 13 X 14 X 15 X 16 IX 17 X 18 WX 19 X 20 (SEQ ID NO: 135); X 13 is A or V; X 14 is S or G; X 15 is Q, E or R; X 16 is G, S, D or N; X 17 is G, S or N; X 18 is S, I, R, T, K, P, N, H or V; X 19 is L or V; X 20 is G or A; The sequence of CDR2L comprises the sequence X 21 AX 22 X 23 X 24 X 25 X 26comprising (SEQ ID NO: 136); X 21 is S, D, E or A; X 22 is S or K; X 23 is S, N, T, R or D; X 24 is L or V; X 25 is Q, E or H; X 26 is S, N, A, R, P or T; The sequence of CDR3L is the sequence QQX 27 X 28 SFPX 29 X 30 comprising (SEQ ID NO: 137); X 27 is A or G; X 28 is N, D or Y; X 29 is F or L; X 30 is A or T.

[0105] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO: 96; The sequence of CDR2L comprises the sequence of SEQ ID NO: 97; The sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

[0106] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; The light chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 98.

[0107] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOs: 21, 22 and 23, respectively; The sequences of CDR1L, CDR2L and CDR3L are SEQ ID NOs: 45, 46 and 47; SEQ ID NOs: 66, 67 and 68; SEQ ID NOs: 70, 67 and 71; SEQ ID NOs: 73, 74 and 75; SEQ ID NOs: 77, 78 and 47; SEQ ID NOs: 80, 81 and 82; SEQ ID NOs: 77, 78 and 84; SEQ ID NOs: 77, 86 and 47; SEQ ID NOs: 88, 89 and 47; SEQ ID NOs: 66, 67 and 47; SEQ ID NOs: 80, 92 and 75; SEQ ID NOs: 80, 94 and 71; SEQ ID NOs: 99, 100 and 47; SEQ ID NOs: 102, 103 and 104; SEQ ID NOs: 106, 103 and 47; SEQ ID NOs: 108, 103 and 47; SEQ ID NOs: 110, 111 and 75; SEQ ID NOs: 77, 103 and 113; SEQ ID NOs: 77, 111 and 47; SEQ ID NOs: 116, 67 and 47; SEQ ID NOs: 118, 119 and 47; SEQ ID NOs: 80, 78 and 47; SEQ ID NOs: 122, 103 and 47; SEQ ID NOs: 124, 125 and 75; SEQ ID NOs: 127, 38 and 68; SEQ ID NOs: 129, 130 and 47 or SEQ ID NOs: 132, 133 and 75.

[0108] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; The light chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134 or any one of SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134.

[0109] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence RSSX1SLLX2SNGX3X4YLD (SEQ ID NO: 62), wherein X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L comprises the sequence X5X6SX7X8X9X 10 (SEQ ID NO: 63), wherein X5 is L, Q or E; X6 is S, A or V; X7 is H, N, T or S; X8 is R or L; X9 is G, A or H; X 10 is S or T; The sequence of CDR3L is the sequence MQGX 11 X 12 contains WPYT (SEQ ID NO: 64), X 11 is A, T, or S; X 12 is H or R.

[0110] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence of SEQ ID NO: 41; The sequence of CDR2L comprises the sequence of SEQ ID NO: 49; The sequence of CDR3L comprises the sequence of SEQ ID NO: 50.

[0111] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: the heavy chain variable region comprises the sequence of SEQ ID NO: 20 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20; the light chain variable region comprises the sequence of SEQ ID NO: 51 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 51.

[0112] In an embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and the CDRs of the heavy and light chain variable regions comprise the following sequences: The sequences of CDR1H, CDR2H, and CDR3H each comprise SEQ ID NOs: 17, 18, and 19, respectively; The sequences of CDR1L, CDR2L, and CDR3L include SEQ ID NO: 41, 42, and 43; SEQ ID NO: 41, 52, and 53; SEQ ID NO: 41, 55, and 56; or SEQ ID NO: 58, 59, and 60, respectively.

[0113] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20; The light chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 44, 54, 57 or 61.

[0114] Fusion proteins comprising the sequences described in Tables 24-27 are provided herein.

[0115] Fusion proteins comprising a heavy chain comprising a sequence selected from the group consisting of SEQ ID NO: 174-184, 187-190, 193-199, 206-211, 224-243 are provided herein. Fusion proteins comprising a light chain comprising a sequence selected from the group consisting of SEQ ID NO: 170, 173, 186, 192 and 205 are provided herein.

[0116] Fusion proteins comprising the light chains and heavy / heavy chain combinations listed in Table 26 are provided herein.

[0117] Fusion proteins comprising the light chains and one or more heavy chains listed in Table 27 are provided herein.

[0118] In an embodiment, a light chain sequence comprising SEQ ID NO: 219 and / or SEQ ID NO: 98 and: (1) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 224; (2) A first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 225; (3) A first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 226; (4) A first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 227 A fusion protein comprising is provided.

[0119] In an embodiment, a light chain sequence comprising SEQ ID NO: 219 and / or SEQ ID NO: 98 and: (1) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 228; (2) A first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 229; (3) A first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 230; (4) A first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 231; (5) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 236; (6) A first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 237; (7) A first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 238; (8) A first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 239; (9) A first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 241; or (10) A first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 242 A fusion protein comprising is provided.

[0120] In an embodiment, a light chain sequence comprising SEQ ID NO: 219 and / or SEQ ID NO: 98 and: (1) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 232; (2) A first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 233; (3) A first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 234; (4) A first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 235; or (5) A first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 243 A fusion protein comprising is provided.

[0121] In an embodiment, a light chain sequence comprising SEQ ID NO: 219 and / or SEQ ID NO: 98 and: (1) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 224; or a first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 225; or (2) A first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 226; or a first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 227 A fusion protein comprising is provided.

[0122] In an embodiment, a light chain sequence comprising SEQ ID NO: 219 and / or SEQ ID NO: 98 and: (1) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 228; or a first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 229; (2) A first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 230; or a first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 231; (3) A first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 236; or a first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 237; (4) a first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 238; or a first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 239; (5) a first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 241; or (6) a first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 242 A fusion protein comprising the same is provided.

[0123] In an embodiment, a light chain sequence comprising SEQ ID NO: 219 and / or SEQ ID NO: 98 and: (1) a first heavy chain sequence comprising SEQ ID NO: 220 and a second heavy chain sequence comprising SEQ ID NO: 232; or a first heavy chain sequence comprising SEQ ID NO: 221 and a second heavy chain sequence comprising SEQ ID NO: 233; (2) a first heavy chain sequence comprising SEQ ID NO: 222 and a second heavy chain sequence comprising SEQ ID NO: 234; or a first heavy chain sequence comprising SEQ ID NO: 223 and a second heavy chain sequence comprising SEQ ID NO: 235; or (3) a first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 243 A fusion protein comprising the same is provided.

[0124] Antibodies and antigen-binding fragments thereof The term "antibody" is used herein in its broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, bispecific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies and grafted antibodies (grafted antibodies), bispecific antibodies, specific antibody portions (e.g., domain antibodies) and any antigen-binding portion thereof that competes with an intact antibody for specific binding, that antigen-binding portion (e.g., paratope, CDR) and any other modified conformation of an immunoglobulin molecule that contains an antigen recognition site that exhibits the desired biological activity and specificity. Thus, an antibody includes an immunoglobulin molecule or a fragment or derivative thereof that contains an antigen-binding site and that can specifically bind to a target by at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The disclosed antibodies can be of mouse, rat, human or any other origin (including chimeric or humanized antibodies).

[0125] In certain embodiments, the framework region of the antibody (or antigen-binding fragment thereof) may be identical to a human germline sequence or may be naturally or artificially modified.

[0126] In one preferred embodiment, the disclosed antibody structure belongs to the IgG class of immunoglobulin molecules. A standard IgG immunoglobulin molecule comprises two identical light chain polypeptides and two identical heavy chain polypeptides. The molecular weight of the light chain polypeptide is approximately 23,000 daltons and the molecular weight of the heavy chain polypeptide varies between 53,000 and 70,000 daltons. The four chains are usually connected by disulfide bonds in a "Y" configuration.

[0127] ​When immunoglobulins are produced by any of hybridomas, B cells or genetically engineered host cells, the two heavy chains (HC) and two light chains (LC) of the immunoglobulin molecule are covalently linked to each other, and the terminal portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent linkages. Both the light and heavy chains contain regions of structural and functional homology. The terms "variable" and "constant" are used functionally. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (composed of domains CH1, CH2 and CH3). Each light chain is composed of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0128] The variable regions enable the antibody to recognize and specifically bind to an epitope located on the antigen. Antigen recognition and specificity are determined by the variable domains of both the light (VL) and heavy (VH) chain portions. The antigen binding site of an antibody is composed of the VL domain and the VH domain or a subset of the CDRs. More specifically, the antigen binding site is defined by one, two or three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) on each of the VH and VL chains. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become distal from the antigen binding site or the amino terminus of the antibody.

[0129] As used herein, the term "complementary determining region" (CDR) refers to the portion of the antibody variable domain that (usually) is involved in antigen binding. Each variable region has three non - contiguous CDRs known as CDR1, CDR2, and CDR3. The CDRs are separated by structurally conserved regions (FR - 1, - 2, - 3, and - 4), called framework regions, which form a "core" β - sheet structure presenting these loops on the surface of the variable domain. The six CDRs present in each antigen - binding domain are short, non - contiguous sequences of amino acids that are specifically arranged to form the antigen - binding domain when the antibody assumes its three - dimensional conformation in an aqueous environment. The length and composition of the CDR sequences are highly variable, particularly in CDR3. The remaining amino acids located in the antigen - binding domain or "framework" regions exhibit less intermolecular variability. The antigen - binding domain formed by the arranged CDRs defines a surface that is complementary to an epitope on the immunoreactive antigen. This complementary surface facilitates the non - covalent binding of the antibody to its cognate epitope. Each CDR can include amino acid residues derived from CDRs as defined, for example, by Kabat (i.e., residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) in the light - chain variable domain and 31 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) in the heavy - chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991)). Each CDR can also include amino acid residues derived from "hypervariable loops" (i.e., approximately residues in the light - chain variable Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (Chothia & Lesk 196 J. Mol. Biol. 901 (1987)) may be included. In some examples, the CDRs may include amino acids from both CDR regions defined according to Kabat and the hypervariable loops. Kabat numbering does not always correspond to the linear numbering in amino acid residues due to shortening or insertion into the structural components, whether it is the framework of the basic variable domain structure or the CDRs. The correct Kabat numbering of the residues of a given antibody or its antigen-binding fragment can be determined by aligning the homologous residues in the sequence of the antibody or its antigen-binding fragment with the "standard" Kabat numbering sequence, or can be defined according to the ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, pp. 55-77 (2003)).

[0130] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" can include Fab, Fab’, F(ab’)2, Fd, Fv, domain antibodies (dAbs such as shark and camel antibodies), ScFv, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv or fragments containing at least certain portions of immunoglobulins sufficient to confer specific antigen binding to a polypeptide.

[0131] The antibody may be an antibody of any class, such as IgG, IgA or IgM (or its subclasses), and the antibody need not be of any particular class, and any immunoglobulin molecule containing an antigen recognition site of the required specificity, other modified conformations (including glycosylation variants of the antibody, amino acid sequence variants of the antibody and antibodies modified by covalent bonds) are included. Modified versions of each of these classes and isotypes are known to those skilled in the art and are thus within the scope of the present disclosure.

[0132] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is a Fab fragment comprising, or consisting essentially of, the variable (VL) and constant (CL) domains of the light chain and the variable domain (VH) and the first constant domain (CH1) of the heavy chain.

[0133] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is a Fab’ fragment referring to a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain.

[0134] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is an Fd fragment comprising, or consisting essentially of, the VH and CH1 domains.

[0135] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is an Fd’ fragment comprising the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain.

[0136] Single-chain Fv or scFv antibody fragments contain, or consist essentially of, the VH and VL domains of an antibody such that these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form the desired structure for antigen binding. Thus, in some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is an Fv fragment that contains, or consists essentially of, the VL and VH domains of a single arm of the antibody.

[0137] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is a diabody that contains two antigen-binding sites comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain.

[0138] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is a dAb fragment that contains, or consists essentially of, the VH domain.

[0139] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is an F(ab’)2 fragment that contains a bivalent fragment that contains two Fab’ fragments linked by a disulfide bridge in the hinge region.

[0140] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is a linear antibody that, together with a complementary light-chain polypeptide, contains a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions.

[0141] One of ordinary skill in the art can use a variety of techniques that have been developed and are available for the generation of antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies. However, F(ab’)2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for generating antibody fragments will be apparent to one of ordinary skill in the art. In other embodiments, the optimal antibody fragment is a single-chain Fv fragment (scFv). See, for example, WO93 / 16185. Alternatively, these fragments can also be generated directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed herein. In another approach, Fab’-SH fragments can be recovered directly from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al., 1992).

[0142] In one embodiment, the antibody is a bispecific antibody that includes a complementarity region that binds to PD-1.

[0143] The contemplated antibodies or antigen-binding fragments can have any isotype, including all classes of constant regions, including IgM, IgG, IgD, and IgE, and any isotype including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype is human IgG1. In another embodiment, human isotype IgG4 is used. The light chain constant region can be λ or κ. The antibody or its antigen-binding fragment can include sequences from more than one class or isotype.

[0144] Anti-PD-1 antibodies and their PD-1 binding fragments The present disclosure describes antibodies that bind to PD-1 and antigen-binding fragments thereof that bind to PD-1, as well as fusion proteins comprising such anti-PD-1 antibodies or antigen-binding fragments thereof. The term "PD-1" refers to the programmed death-1 protein (also known as CD279), a T cell co-inhibitor. The term PD-1 encompasses recombinant PD-1 and / or fragments thereof. The term also includes, for example, PD-1 or fragments thereof coupled to mouse or human Fc, histidine tags and / or signal sequences. The term may further encompass fusion proteins comprising PD-1. The amino acid sequence of full-length PD-1 is provided in GenBank under accession number NP_005009.2. Ligands of PD-1 include PD-L1 and PD-L2. The amino acid sequence of full-length PD-L1 is provided in GenBank under accession number NP_054862.1. The amino acid sequence of full-length PD-L2 is provided in GenBank under accession number NP_079515.2.

[0145] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof specifically binds to PD-1 and antagonizes PD-1-mediated immunosuppression. The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein may prevent, inhibit, and reduce PD-1 biological activity, including downstream events mediated by PD-1. The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein may exhibit one or more of the following functions: (a) binding to PD-1 and blocking downstream signaling events; (b) blocking PD-L1-binding to PD-1; (c) increasing T cell proliferation; (d) upregulating T cell-mediated immune responses; (e) stimulating TNF secretion; (f) reducing inhibitory signaling by PD-1; and / or (g) stimulating IFNγ secretion. The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein exhibit strong binding and inhibitory activity and are useful for therapeutic and diagnostic uses.

[0146] In one aspect, the present disclosure provides an antibody that binds to PD-1 and antigen-binding fragments thereof. In certain embodiments, the present disclosure provides bispecific antibodies and binding proteins that specifically bind to PD-1 and at least one other molecule.

[0147] In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof binds to PD-1, the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region, each of the heavy-chain and light-chain variable regions comprises CDR1, CDR2, and CDR3, and the antibody heavy chain comprises a constant region comprising three constant domains CH1, CH2, and CH3.

[0148] In embodiments, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence RX 13 X 14 X 15 X 16 IX 17 X 18 WX 19 X 20 (SEQ ID NO: 135); X 13 is A or V; X 14 is S or G; X 15 is Q, E, or R; X 16 is G, S, D, or N; X 17 is G, S, or N; X 18 is S, I, R, T, K, P, N, H, or V; X 19 is L or V; X 20 is G or A; The sequence of CDR2L comprises the sequence X21 AX 22 X 23 X 24 X 25 X 26 (SEQ ID NO: 136); X 21 is S, D, E or A; X 22 is S or K; X 23 is S, N, T, R or D; X 24 is L or V; X 25 is Q, E or H; X 26 is S, N, A, R, P or T; The sequence of CDR3L is the sequence QQX 27 X 28 SFPX 29 X 30 (SEQ ID NO: 137); X 27 is A or G; X 28 is N, D or Y; X 29 is F or L; X 30 is A or T.

[0149] In an embodiment, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO: 96; The sequence of CDR2L comprises the sequence of SEQ ID NO: 97; The sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

[0150] In an embodiment, the antibody or antigen-binding fragment thereof is as follows: A heavy chain variable region containing SEQ ID NO: 24 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; and A light chain variable region containing SEQ ID NO: 98 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 98 is included.

[0151] In an embodiment, the CDRs of the anti-PD-1 antibody or an antigen-binding fragment thereof comprise the following sequences: The sequences of CDR1H, CDR2H and CDR3H each comprise SEQ ID NO: 21, 22 and 23 respectively; The sequences of CDR1L, CDR2L and CDR3L are SEQ ID NO: 45, 46 and 47 respectively; SEQ ID NO: 66, 67 and 68; SEQ ID NO: 70, 67 and 71; SEQ ID NO: 73, 74 and 75; SEQ ID NO: 77, 78 and 47; SEQ ID NO: 80, 81 and 82; SEQ ID NO: 77, 78 and 84; SEQ ID NO: 77, 86 and 47; SEQ ID NO: 88, 89 and 47; SEQ ID NO: 66, 67 and 47; SEQ ID NO: 80, 92 and 75; SEQ ID NO: 80, 94 and 71; SEQ ID NO: 99, 100 and 47; SEQ ID NO: 102, 103 and 104; SEQ ID NO: 106, 103 and 47; SEQ ID NO: 108, 103 and 47; SEQ ID NO: 110, 111 and 75; SEQ ID NO: 77, 103 and 113; SEQ ID NO: 77, 111 and 47; SEQ ID NO: 116, 67 and 47; SEQ ID NO: 118, 119 and 47; SEQ ID NO: 80, 78 and 47; SEQ ID NO: 122, 103 and 47; SEQ ID NO: 124, 125 and 75; SEQ ID NO: 127, 38 and 68; SEQ ID NO: 129, 130 and 47; or SEQ ID NO: 132, 133 and 75.

[0152] In an embodiment, the anti-PD-1 antibody or an antigen-binding fragment thereof is as follows: A heavy chain variable region containing SEQ ID NO: 24 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24; and A light chain variable region containing SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134 is included.

[0153] In an embodiment, the CDRs of the anti-PD-1 antibody or its antigen-binding fragment comprise the following sequences: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence RSSX1SLLX2SNGX3X4YLD (SEQ ID NO: 62), and X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L comprises the sequence X5X6SX7X8X9X 10 (SEQ ID NO: 63), and X5 is L, Q or E; X6 is S, A or V; X7 is H, N, T or S; X8 is R or L; X9 is G, A or H; X 10 is S or T; The sequence of CDR3L is the sequence MQGX 11 X 12 includes WPYT (SEQ ID NO: 64), X 11 is A, T, or S; X 12 is H or R.

[0154] In an embodiment, the CDRs of the anti-PD-1 antibody or an antigen-binding fragment thereof include the following sequences: The sequence of CDR1H includes the sequence of SEQ ID NO: 17; The sequence of CDR2H includes the sequence of SEQ ID NO: 18; The sequence of CDR3H includes the sequence of SEQ ID NO: 19; The sequence of CDR1L includes the sequence of SEQ ID NO: 41; The sequence of CDR2L includes the sequence of SEQ ID NO: 49; The sequence of CDR3L includes the sequence of SEQ ID NO: 50.

[0155] In an embodiment, the anti-PD-1 antibody or an antigen-binding fragment thereof is as follows: a heavy chain variable region comprising SEQ ID NO: 20 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20; and a light chain variable region comprising SEQ ID NO: 51 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 including.

[0156] In an embodiment, the CDRs of the anti-PD-1 antibody or an antigen-binding fragment thereof include the following sequences: The sequences of CDR1H, CDR2H, and CDR3H each include SEQ ID NOs: 17, 18, and 19, respectively; The sequences of CDR1L, CDR2L, and CDR3L each include SEQ ID NOs: 41, 42, and 43; SEQ ID NOs: 41, 52, and 53; SEQ ID NOs: 41, 55, and 56; or SEQ ID NOs: 58, 59, and 60.

[0157] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1 and the antibody or antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region, and the sequence of the heavy-chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20 and 24, and the sequence of the light-chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 32, 36, 40, 44, 48, 51, 54, 57, 61, 65, 69, 72, 76, 79, 83, 85, 88, 90, 91, 93, 95, 98, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131, 134 and 138.

[0158] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, and the antibody or antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region, and the sequence of the heavy-chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 20, and the sequence of the light-chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 65.

[0159] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 20. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region selected from the group consisting of SEQ ID NOs: 44, 51, 54, 57 and 61.

[0160] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 20. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 51.

[0161] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 20. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 54.

[0162] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 20. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 57. and includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 57.

[0163] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 20. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 61.

[0164] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 138.

[0165] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 20. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region selected from the group consisting of SEQ ID NOs: 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 98, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131, 134 and 138.

[0166] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 98.

[0167] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 69.

[0168] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 72.

[0169] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 76.

[0170] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 79.

[0171] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 83.

[0172] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 85.

[0173] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 87.

[0174] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 90.

[0175] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 91.

[0176] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 93.

[0177] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 95.

[0178] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 101.

[0179] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 105.

[0180] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 107.

[0181] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 109.

[0182] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 112.

[0183] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 114.

[0184] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 115.

[0185] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 117.

[0186] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 120.

[0187] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region the sequence of which comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 121.

[0188] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 123.

[0189] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 126.

[0190] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, the sequence of the heavy-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy-chain variable region amino acid sequence of SEQ ID NO: 24, and the sequence of the light-chain variable region comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light-chain variable region amino acid sequence of SEQ ID NO: 128.

[0191] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 131.

[0192] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 24. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region of SEQ ID NO: 134.

[0193] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1. The antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region. The sequence of the heavy-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 142. The sequence of the light-chain variable region includes a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light-chain variable region selected from the group consisting of SEQ ID NOs: 146, 150, 153, 156, 160, 164 and 167.

[0194] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, each of the heavy-chain and light-chain variable regions comprising CDR1, CDR2, and CDR3, where: the sequence of CDR1H comprises the sequence of SEQ ID NO: 139; the sequence of CDR2H comprises the sequence of SEQ ID NO: 140; the sequence of CDR3H comprises the sequence of SEQ ID NO: 141; the sequence of CDR1L comprises the sequence of SEQ ID NO: 143; the sequence of CDR2L comprises the sequence of SEQ ID NO: 144; the sequence of CDR3L comprises the sequence of SEQ ID NO: 145.

[0195] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, each of the heavy-chain and light-chain variable regions comprising CDR1, CDR2, and CDR3, where: the sequence of CDR1H comprises the sequence of SEQ ID NO: 139; the sequence of CDR2H comprises the sequence of SEQ ID NO: 140; the sequence of CDR3H comprises the sequence of SEQ ID NO: 141; the sequence of CDR1L comprises the sequence of SEQ ID NO: 147; the sequence of CDR2L comprises the sequence of SEQ ID NO: 148; the sequence of CDR3L comprises the sequence of SEQ ID NO: 149.

[0196] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, each of the heavy-chain and light-chain variable regions comprising CDR1, CDR2, and CDR3, where: the sequence of CDR1H comprises the sequence of SEQ ID NO: 139; the sequence of CDR2H comprises the sequence of SEQ ID NO: 140; the sequence of CDR3H comprises the sequence of SEQ ID NO: 141; The sequence of CDR1L includes the sequence, SEQ ID NO: 151; The sequence of CDR2L includes the sequence, SEQ ID NO: 148; The sequence of CDR3L includes the sequence, SEQ ID NO: 152.

[0197] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region, and each of the heavy-chain and light-chain variable regions includes CDR1, CDR2, and CDR3, where: The sequence of CDR1H includes the sequence, SEQ ID NO: 139; The sequence of CDR2H includes the sequence, SEQ ID NO: 140; The sequence of CDR3H includes the sequence, SEQ ID NO: 141; The sequence of CDR1L includes the sequence, SEQ ID NO: 154; The sequence of CDR2L includes the sequence, SEQ ID NO: 148; The sequence of CDR3L includes the sequence, SEQ ID NO: 155.

[0198] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region, and each of the heavy-chain and light-chain variable regions includes CDR1, CDR2, and CDR3, where: The sequence of CDR1H includes the sequence, SEQ ID NO: 139; The sequence of CDR2H includes the sequence, SEQ ID NO: 140; The sequence of CDR3H includes the sequence, SEQ ID NO: 141; The sequence of CDR1L includes the sequence, SEQ ID NO: 157; The sequence of CDR2L includes the sequence, SEQ ID NO: 158; The sequence of CDR3L includes the sequence, SEQ ID NO: 159.

[0199] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region, and each of the heavy-chain and light-chain variable regions includes CDR1, CDR2, and CDR3, where: The sequence of CDR1H includes the sequence, SEQ ID NO: 139; The sequence of CDR2H includes the sequence, SEQ ID NO: 140; The sequence of CDR3H includes the sequence, SEQ ID NO: 141; The sequence of CDR1L includes the sequence, SEQ ID NO: 161; The sequence of CDR2L includes the sequence, SEQ ID NO: 162; The sequence of CDR3L includes the sequence, SEQ ID NO: 163.

[0200] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to PD-1, the antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region, each of the heavy-chain and light-chain variable regions comprising CDR1, CDR2, and CDR3, wherein: The sequence of CDR1H includes the sequence, SEQ ID NO: 139; The sequence of CDR2H includes the sequence, SEQ ID NO: 140; The sequence of CDR3H includes the sequence, SEQ ID NO: 141; The sequence of CDR1L includes the sequence, SEQ ID NO: 157; The sequence of CDR2L includes the sequence, SEQ ID NO: 165; The sequence of CDR3L includes the sequence, SEQ ID NO: 166.

[0201] In addition to the above description, FIG. 1 shows the heavy- and light-chain variable regions and related CDRs of the antibodies disclosed herein, similar to the accompanying sequence listing incorporated herein by reference in its entirety. As shown in the following examples, light-chain shuffling of the anti-PD-1 antibody led to the identification of antibody variants that gave a substantial improvement in PD-1 binding affinity. Analysis of the variants revealed certain CDR positions that remained relatively invariant among antibodies with light-chain shuffling of the amino acids, and other CDR positions where variation could be introduced without abolishing PD-1 binding.

[0202] The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein, as well as the fusion proteins disclosed herein, may have one or more amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, one or more CDR residues of the anti-PD-1 antibody or antigen-binding fragment (or fusion protein comprising an anti-PD-1 antibody or antigen-binding fragment) disclosed herein are altered by amino acid substitution, deletion, insertion, and / or addition. The amino acid substitutions may be conservative substitutions or non-conservative substitutions. The present disclosure also encompasses anti-PD-1 antibodies and antigen-binding fragments thereof (and fusion proteins comprising an anti-PD-1 antibody or antigen-binding fragment) that are derived from the amino acid sequences disclosed herein and in which one or more amino acids within one or more framework and / or CDR regions have been mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). In certain embodiments, the anti-PD-1 antibody or its binding fragment (or fusion protein comprising an anti-PD-1 antibody or antigen-binding fragment) has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the CDR and / or variable domain sequences shown in FIG. 1, and comprises one or more CDRs, or one or more variable domains.

[0203] Also provided herein are variable heavy and variable light chain sequences and pairings thereof that are similar but not identical to those disclosed in Table 25. It will be apparent that any of the frameworks described herein can be utilized in combination with any of the CDRs and CDR motifs described herein. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment utilizes the framework described in Table 25.

[0204] Also provided herein are chimeric antigen receptors (CARs) comprising 1, 2, 3, 4, 5, or 6 CDRs of the anti-PD1 antibodies and antigen-binding fragments disclosed herein. Also provided herein are CARs comprising 6 CDRs of any one of the anti-PD1 antibodies and antigen-binding fragments disclosed herein.

[0205] Disclosed herein are immune cells expressing a CAR comprising 1, 2, 3, 4, 5, or 6 CDRs of the anti-PD1 antibodies and antigen-binding fragments disclosed herein. Disclosed herein are immune cells expressing a CAR comprising 6 CDRs of any one of the anti-PD1 antibodies and antigen-binding fragments disclosed herein. In some embodiments, the immune cell is a T cell.

[0206] "Identity" refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences in an optimally aligned sequence, after consideration of the number of gaps required for optimal alignment and the length of each such gap. "Substantially identical" means an amino acid sequence that differs from the original sequence only by conservative amino acid substitutions and that does not disrupt the function of the protein.

[0207] Also disclosed herein are anti-PD-1 antibodies or antigen-binding fragments or fusion proteins thereof that comprise an amino acid sequence that is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% or at least 99% identical to the amino acid sequences disclosed herein. Among other things, methods and computer programs for determining sequence similarity, including the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990) and the ALIGN program (version 2.0) are publicly available. The Smith Waterman algorithm can also be used to determine similarity. The BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Maryland 20894; BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). In comparing sequences, these methods account for various substitutions, deletions and other modifications.

[0208] In some embodiments of aspects described herein, amino acid sequence modifications of antibodies or antigen-binding fragments thereof that bind to PD-1 or fusion proteins that bind to PD-1 described herein are contemplated. Amino acid sequence variants of the anti-PD-1 antibody or antigen-binding fragment or fusion protein are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the anti-PD-1 antibody or antigen-binding fragment or fusion protein or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding fragment thereof. Any combination of deletions, insertions and substitutions may be made to reach the final construct, provided that the final construct has the desired characteristics, such as binding specificity, inhibition of biological activity. Combination is made.

[0209] In some cases, amino acid substitutions can be made by selecting substitutions that do not significantly differ in (a) the structure of the peptide backbone in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site; or (c) its effect on maintaining the bulk of the side chain (conservative amino acid substitution variants). These variants have at least one amino acid residue in an antibody or an antigen-binding fragment or fusion protein thereof that has been replaced by a different residue having similar side chain properties. Amino acids can be grouped according to the similarity of their side chain properties (see Lehninger, BIOCHEMISTRY (2nd Edition, Worth Publishers, New York, 1975): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H).

[0210] As such, non-limiting examples of conservative amino acid substitutions are those that replace a nonpolar amino acid with another nonpolar amino acid.

[0211] Alternatively, naturally occurring residues can be grouped into groups based on common side chain properties: (1) Hydrophobic: Ala (A), Val (V), Leu (L), Ile (I), Met (M); (2) Neutral hydrophilic: Ser (S), Thr (T), Cys (C), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H); (5) Residues affecting chain orientation: Gly (G), Pro (P); (6) Aromatic: Phe (F), Trp (W), Tyr (Y).

[0212] Substitutions made within these groups can be considered conservative substitutions. Examples of non-limiting substitutions include substitution of valine with alanine, lysine with arginine, glutamine with asparagine, glutamic acid with aspartic acid, serine with cysteine, asparagine with glutamine, aspartic acid with glutamic acid, proline with glycine, arginine with histidine, leucine with isoleucine, isoleucine with leucine, arginine with lysine, leucine with methionine, leucine with phenylalanine, glycine with proline, threonine with serine, serine with threonine, tyrosine with tryptophan, phenylalanine with tyrosine, and / or leucine with valine.

[0213] Also contemplated are amino acid sequence insertions that can include amino-terminal and / or carboxyl-terminal fusions and insertions within the sequence of single or multiple amino acid residues of a polypeptide length range containing from 1 residue to 100 or more residues. Examples of terminal fusions include an antibody or antigen-binding fragment thereof having an N-terminal methionyl residue or an antibody or antigen-binding fragment thereof fused to a cytotoxic polypeptide (or a fusion protein containing such an antibody or antigen-binding fragment thereof). Other examples of terminal fusions of an antibody or antigen-binding fragment thereof include, for example, fusions of an antibody or antigen-binding fragment thereof to an enzyme or polypeptide that increases the serum half-life of the antibody or antigen-binding fragment thereof, such as biotin, to the N-terminal or C-terminal of the antibody or antigen-binding fragment thereof (or a fusion protein containing such an antibody or antigen-binding fragment thereof) are included.

[0214] To improve the oxidative stability of the molecule and prevent abnormal cross-linking, any cysteine residues not involved in maintaining the proper conformation of an antibody or antigen-binding fragment thereof that binds to PD-1 can also be substituted, for example, with serine or alanine.

[0215] Conversely, a cysteine bond can be added to an anti-PD-1 antibody or an antigen-binding fragment thereof to improve its stability (particularly when the anti-PD-1 antibody or an antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).

[0216] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof, or a fusion protein comprising the described antibody or an antigen-binding fragment thereof, has an amino acid change that alters the original glycosylation pattern of the anti-PD-1 antibody or an antigen-binding fragment thereof. "Altering the original glycosylation pattern" means deleting one or more carbohydrate moieties found in the antibody or an antigen-binding fragment thereof and / or adding one or more glycosylation sites not present in the antibody or an antigen-binding fragment thereof. Glycosylation of an antibody is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide results in a potential glycosylation site. O-linked glycosylation refers to the attachment of one of galactose N-acetylgalactosamine, galactose or xylose, which can also use 5-hydroxyproline or 5-hydroxylysine, but most commonly serine or threonine, to a hydroxyamino acid, most commonly serine or threonine. The alteration can also be done by the addition or substitution of one or more serine or threonine residues to the sequence of the original antibody or an antigen-binding fragment thereof (for O-linked glycosylation sites).

[0217] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof may include modifications including, but not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization, etc., by known protecting groups / blocking groups, proteolytic cleavage, ligation to a cellular ligand or other protein, etc. The process of chemical modification is known in the art and includes, but is not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the molecule may contain one or more non-classical amino acids.

[0218] The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein may include anti-PD-1 antibodies and antigen-binding fragments thereof whose binding characteristics have been altered by direct mutagenesis, affinity maturation, phage display, or chain shuffling. Affinity and specificity are altered by mutating the CDRs and screening for CDRs with desired characteristics. Methods of mutagenesis are known to those of skill in the art.

[0219] Antibody binding Also provided herein are fusion proteins comprising an anti-PD-1 antibody and antigen-binding fragment thereof, and an anti-PD-1 antibody or antigen-binding fragment thereof that binds to an epitope on PD-1 that is identical to one of the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein.

[0220] In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein selectively bind to PD-1 over one or more PD family members. It binds. In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment does not show significant binding to other PD family members, including binding to CTLA-4 and / or CD28. The human PD-1 gene produces four alternatively spliced PD-1 mRNA transcripts. One of these variants produces a soluble form of PD-1. In one aspect, the present disclosure provides an anti-PD-1 antibody or its antigen-binding fragment that binds to all isoforms of PD-1. In some embodiments, the provided anti-PD-1 antibody and its antigen-binding fragment bind to mammalian PD-1.

[0221] As used herein, "binding" of an antibody or its antigen-binding fragment or a fusion protein comprising an antibody or its antigen-binding fragment to PD-1, an epitope on PD-1, or, in certain embodiments described below, to specific residues on PD-1, includes the selective interaction of the antibody or its antigen-binding fragment with PD-1. Thus, binding includes primary or secondary interactions including, for example, hydrogen bonds, ionic interactions, salt bridges, and hydrophilic and hydrophobic interactions.

[0222] In certain embodiments, the anti-PD-1 antibody or its antigen-binding fragment described herein binds to PD-1 with an equilibrium dissociation (KD) constant of 10 -2 ~10 -10 mol / l, 10 -3 ~10 -10 mol / l, 10 -4 ~10 -10 mol / l, 10 -5 ~10 -10 mol / l, 10 -6 ~10 -10 mol / l, 10 -7 ~10 -10 mol / l, 10 -8 ~10 -10 mol / l or 10 -9 ~10 -10 mol / l. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment described herein binds to PD-1 with a KD of 10 -2 ~10 -9mol / l, 10 -3 ~10 -9 mol / l, 10 -4 ~10 -9 mol / l, 10 -5 ~10 -9 mol / l, 10 -6 ~10 -9 mol / l, 10 -7 ~10 -9 mol / l, 10 -8 ~10 -9 mol / l or 10 -9 ~10 -10 binds with a KD of 10 mol / l or 10~10 mol / l.

[0223] As used herein, "affinity," represented by the KD of an antigen for its antigen-binding protein, is a measure of the strength of binding between an antigenic determinant and an antigen-binding site on an antigen-binding protein such as an antibody or an antibody fragment thereof. The K D value is inversely proportional to the strength of binding between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can also be expressed as the association constant (K D ) which is 1 / K A . Affinity can be determined by those skilled in the art in a manner known per se depending on the specific antigen of interest.

[0224] The term "specificity" as used herein refers to the ability of an antibody or an antigen-binding fragment thereof, such as an anti-PD-1 antibody or an antigen-binding fragment thereof, to recognize an epitope within PD-1 while having little or no detectable reactivity with other portions of PD-1. Specificity can be determined relatively by a competitive assay or by an epitope identification / characterization technique described herein or its equivalents known in the art.

[0225] As used herein, the term "epitope" refers to a specific target to which an antibody binds. Epitopes are formed by both a continuous stretch of amino acids (continuous epitope) and a three-dimensional arrangement of amino acid residues (non-continuous epitope) that exist only when the target protein is folded into a specific conformation. Generally, an epitope comprises at least 3 amino acids, at least 4, at least 5, or about 7-10 amino acids.

[0226] Disclosed herein are anti-PD-1 antibodies and antigen-binding fragments thereof, and fusion proteins comprising an anti-PD1 antibody or an antigen-binding fragment thereof that specifically bind to the same epitope as anti-PD-1 antibody 38B2. Also, anti-PD-1 antibodies and antigen-binding fragments thereof, and fusion proteins comprising an anti-PD1 antibody or an antigen binding fragment thereof that bind to the same epitope as anti-PD-1 antibody 31B1 are disclosed herein.

[0227] As used herein, a "blocking" antibody or antibody "antagonist" is one that inhibits or reduces the biological activity of the antigen to which it binds. For example, in some embodiments, an anti-PD-1 antagonist antibody or an antigen-binding fragment thereof binds to PD-1 and inhibits the activity of PD-1 and / or the binding of PD-1 to a binding partner such as PD-L1 or PD-L2. Inhibition of activity and inhibition of binding include partial inhibition. Methods for identifying PD-1 antibodies that block PD-1 interactions are described herein and are known to those of skill in the art. For example, competitive antibodies, cross-blocking antibodies, and cross-blocked antibodies can be identified using any suitable method known in the art, including competitive ELISA or BIACORE® assays in which the binding of a competitive antibody or cross-blocking antibody to human PD-1 inhibits the binding of the antibodies disclosed herein, or vice versa.

[0228] In certain embodiments, not all CDRs are directly involved in binding to the antigen. In one embodiment, four of the six CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, five of the six CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, all six of the six CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof contact the antigen.

[0229] As used herein, the terms "selective" and "selectivity" refer to the preferential binding of an antibody or antigen-binding fragment thereof (i.e., a PD-1 antibody or antigen-binding fragment thereof) to a region or epitope in PD-1, as contrasted with a particular region, target or peptide; typically one or more other biomolecules including other PD-1 family members.

[0230] In one aspect, provided are anti-PD-1 antibodies and antigen-binding fragments thereof that specifically bind to at least a portion of a binding site on PD-1, thereby blocking PD-1 interaction with one or more PD-1 ligands. These PD-1 ligands include, but are not limited to, PD-L1 and PD-L2.

[0231] In certain embodiments, an anti-PD-1 antibody or antigen-binding fragment according to the present disclosure includes an Fc domain composed of a first and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains that include the heavy chain domains of an immunoglobulin molecule. The two subunits of the Fc domain form a stable association. In embodiments, the two subunits of the Fc domain are identical. In alternative embodiments, the two subunits of the Fc domain are not identical. In embodiments, one subunit of the Fc domain is fused to an immune complex molecule. In embodiments, the Fc domain of the antibody can be an IgG Fc domain, an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain. In further particular embodiments, the Fc domain is a human Fc domain.

[0232] Fc domain modifications that promote heterodimerization Modifications in the Fc domain of the disclosed anti-PD-1 antibodies or antigen-binding fragments thereof that promote dimerization are further contemplated. In embodiments, the Fc domain of the anti-PD-1 antibody or antigen-binding fragment thereof comprises modifications that promote the association of the first and second subunits of the Fc domain. In one embodiment, the modification is in the CH3 domain of the Fc domain. In certain embodiments, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0233] The knob-into-hole modification is a "protrusion-into-cavity" strategy that serves to manipulate the interface between the first and second polypeptides for hetero-oligomerization. The "protrusion" (i.e., the knob) is constructed by replacing small amino acid side chains derived from the interface of the first polypeptide with larger side chains. Optionally, a compensatory "cavity" (i.e., the hole) of the same or smaller size relative to the protrusion is created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones. In certain embodiments, the amino acid residues in the CH3 domain of the first Fc subunit are replaced with amino acid residues having a larger side chain volume, thereby generating a knob within the CH3 domain of the first Fc subunit, which can be positioned within a hole present within the CH3 domain of the second Fc subunit that is generated by replacing one amino acid residue in the CH3 domain of the second Fc subunit with an amino acid residue having a smaller side chain volume. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of cysteine (C), valine (V), alanine (A), phenylalanine (F), tyrosine (Y), leucine (L), lysine (K), proline (P), glutamic acid (E), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), arginine (R), tryptophan (W), cysteine (C), lysine (L), glutamic acid (E), aspartic acid (D), and valine (V).

[0234] Mutations corresponding to the knob and hole can be made, for example, by site-directed mutagenesis or by peptide synthesis by altering the nucleic acid encoding the polypeptide.

[0235] In embodiments of the fusion protein where only one heavy chain is linked to the IL-15 / IL-15Rα sushi, the Fc domain of the anti-PD-1 antibody or antigen-binding fragment thereof may contain one or more amino acid substitutions that promote heterodimer formation (i.e., association of the heavy chain fusion with the heavy chain lacking the fusion). In embodiments, the amino acid residue in the CH3 domain (CH3-1) of one heavy chain is replaced with an amino acid residue having a larger side chain volume, thereby including an amino acid substitution that creates a "knob" within the CH3 domain, which can be placed within a "hole" present in the CH3 domain (CH3-2) of the other heavy chain generated by replacing the amino acid residue with an amino acid residue having a smaller side chain volume. In embodiments, the CH3 domain of the first subunit ("knob" subunit) of the Fc domain may include one or more substitutions from the following: T350V, L351Y, S354C, S364H, T366Y, T366W, F405A, Y407V. In certain embodiments, the CH3 domain of the second subunit ("hole" subunit) of the Fc domain may include one or more substitutions from the following: Y349C, T350V, T366L, T366S, L368A, K392L, T394W, Y407V, Y407T.

[0236] In some embodiments, the amino acid substitutions are (1) CH3 domain of the first heavy chain: S354C, T366W; CH3 domain of the second heavy chain: Y349C, T366S, L368A, Y407V; (2) CH3 domain of the first heavy chain: T350V, L351Y, F405A, Y407V; CH3 domain of the second heavy chain: T350V, T366L, K392L, T394W; (3) CH3 domain of the first heavy chain: L351Y, F405A, Y407V; CH3 domain of the second heavy chain: T366L, K392L, T394W; (4) CH3 domain of the first heavy chain: T366W; CH3 domain of the second heavy chain: Y407T; (5) CH3 domain of the first heavy chain: T366Y; CH3 domain of the second heavy chain: Y4 07T; (6) CH3 domain of the first heavy chain: T366W; CH3 domain of the second heavy chain: Y407A; (7) CH3 domain of the first heavy chain: F405A; CH3 domain of the second heavy chain: T394W; (8) CH3 domain of the first heavy chain: F405W; CH3 domain of the second heavy chain: T394S; (9) CH3 domain of the first heavy chain: T366Y and F405A; CH3 domain of the second heavy chain: T394W and Y407T; (10) CH3 domain of the first heavy chain: T366W and F405W; CH3 domain of the second heavy chain: T394S: and 407A; (11) CH3 domain of the first heavy chain: F405W and Y407A; CH3 domain of the second heavy chain: T366W and T394S (12) selected from.

[0237] An antibody or antigen-binding fragment thereof (or a fusion protein comprising such an antibody or antigen-binding fragment) that binds to PD-1 is provided herein, and the antibody comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 168-169, 171-172, 185, 191 and 201-203.

[0238] An antibody or antigen-binding fragment thereof (or a fusion protein comprising such an antibody or antigen-binding fragment) that binds to PD-1 is provided herein, and the antibody comprises a light chain comprising a sequence selected from the group consisting of SEQ ID NOs: 170, 173, 186, 192 and 205.

[0239] In certain embodiments, the Fc domain according to the present disclosure exhibits a reduced binding affinity for Fc receptors and / or a reduced effector function as compared to the native IgG1 Fc domain. In certain embodiments, the Fc domain is engineered to have a reduced binding affinity for Fc receptors and / or a reduced effector function as compared to the unengineered Fc domain.

[0240] Linker In embodiments, the fusion proteins provided herein may include one or more linkers that connect the components of the fusion proteins disclosed herein. The linker can be (i) between the IL-15 polypeptide and the IL15Rα polypeptide comprising the sushi domain; (ii) between the heavy chain of the anti-PD-1 antibody or antigen-binding fragment thereof and the IL-15 polypeptide or the IL15Rα polypeptide comprising the sushi domain; or (iii) located at both. In embodiments of the present disclosure, the IL-15 polypeptide and the IL15Rα polypeptide comprising the sushi domain are joined or linked by a first linker amino acid sequence. In embodiments, the IL-15 polypeptide (or IL15Rα polypeptide) is linked to the antibody or antigen-binding fragment thereof described herein by a second linker amino acid sequence. In embodiments, the second linker connects the N-terminus of the heavy chain variable region to the C-terminus of the IL-15 polypeptide. The first and second linkers can have the same or different amino acid sequences.

[0241] The linker amino acid sequences described herein can be of a length sufficient to ensure that the fusion protein forms an appropriate secondary and tertiary structure. The length of the linker amino acids can be between 5 and 40 amino acids, preferably between 10 and 40 amino acids, more preferably between 15 and 40 amino acids, even more preferably between 20 and 40 amino acids, and most preferably between 25 and 35 amino acids.

[0242] Preferably, the linker sequence contains neutrally charged amino acids selected from the group consisting of Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L), and Gln (Q), and most preferably amino acids selected from the group consisting of Gly (G), Asn (N), and Ser (S). Preferably, the linker sequence is glycine-rich and serine-rich, and in some embodiments, the linker contains only serine and glycine residues.

[0243] In some embodiments, the linker includes a portion of the IL15Rα polypeptide outside the sushi domain that includes, but is not limited to, the bolded sequences in the linker of SEQ ID NO: 215 (below).

[0244] Non-limiting examples of linkers for a portion of the amino acid sequence include IRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 215), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 216); IRDPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 217), IRDPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 218), and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 244) are included.

[0245] In embodiments, the linker that connects the IL-15 polypeptide and the IL-15Rα sushi includes IRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 215), IRDPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 217), or IRDPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 218) is included.

[0246] In embodiments, the linker that connects the IL-15 polypeptide to the anti-PD-1 antibody heavy chain includes GGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 216) or GGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 244) is included.

[0247] In an embodiment, the heavy and light chains of the anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein may be connected to a single polypeptide chain ("single-chain Fv" or "scFv") using a third linker that allows the VR and VL domains to associate to form an antigen-binding site. The amino acid sequence of the linker may be the same or different.

[0248] In one embodiment, the IL-15 polypeptide or IL-15 derivative is covalently linked by a linker to the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative.

[0249] In one embodiment, the IL-15 polypeptide or IL-15 derivative is covalently linked by a linker to the anti-PD-1 antibody or antigen-binding fragment thereof. In one embodiment, the C-terminus of the IL-15 polypeptide or the IL-15 derivative is covalently linked by a linker to the N-terminus of the anti-PD-1 antibody or antigen-binding fragment thereof. In one embodiment, the N-terminus of the IL-15 polypeptide or the IL-15 derivative is covalently linked by a linker to the C-terminus of the anti-PD-1 antibody or antigen-binding fragment thereof. In one embodiment, the N-terminus of the IL-15 polypeptide or the IL-15 derivative is covalently linked by a linker to the CH3 region of the anti-PD-1 antibody or antigen-binding fragment thereof.

[0250] In one embodiment, the IL-15Rα sushi or IL-15Rα sushi derivative is covalently linked to an anti-PD-1 antibody or an antigen-binding fragment thereof by a linker. In one embodiment, the C-terminus of the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative is covalently linked to the N-terminus of the anti-PD-1 antibody or an antigen-binding fragment thereof by a linker. In one embodiment, the N-terminus of the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative is covalently linked to the C-terminus of the anti-PD-1 antibody or an antigen-binding fragment thereof by a linker. In one embodiment, the N-terminus of the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative is covalently linked to the CH3 region of the anti-PD-1 antibody or an antigen-binding fragment thereof by a linker.

[0251] In some embodiments, the IL-15 polypeptide or IL-15 derivative is covalently linked to the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative by a first linker, and either the IL-15 polypeptide or IL-15 derivative or the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative is covalently linked to the anti-PD-1 antibody or an antigen-binding fragment thereof by a second linker. In some embodiments, the amino acid sequences of the first and second linkers are the same. In other embodiments, the amino acid sequences of the first and second linkers are different.

[0252] Conjugate The anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein may further comprise one or more functional moieties. Examples of useful functional moieties include, but are not limited to, blocking moieties, detectable moieties, diagnostic moieties, targeting and therapeutic moieties.

[0253] The blocking moiety can include sufficient steric bulk and / or charged moieties such that reduced glycosylation occurs, for example, by blocking the ability of a sialidase to glycosylate an antibody or antigen-binding fragment thereof. Preferred blocking moieties include cysteine, cysteine adducts such as mixed disulfide adducts or disulfide bonds, and PEG moieties such as polyethylene glycol (“PEG”), polypropylene glycol (“PPG”), polyoxyethylated glycerol (“POG”) and other polyoxyethylated polyols, polyvinyl alcohol (“PVA”) and other polyalkylene oxides, polyoxyethylated sorbitol or polyoxyethylated glucose. PEG is a preferred moiety in biological applications for several reasons. PEGylation can improve the pharmacokinetic performance of a molecule by increasing the apparent molecular weight of the molecule. The increased apparent molecular weight reduces the rate of clearance from the body after subcutaneous or systemic administration. In many cases, PEGylation can reduce antigenicity and immunogenicity. PEGylation also increases the solubility of biologically active molecules. Additionally, PEG is typically clear, colorless, odorless, water-soluble, stable to heat, inert to many chemicals, non-hydrolyzing, and non-toxic, making it a preferred choice for biological applications.

[0254] Examples of detectable moieties conjugated to the anti-PD-1 antibodies or antigen-binding fragments or fusions disclosed herein include fluorescent moieties or labels, contrast agents, radioisotope moieties, radiopaque moieties, etc., for example, detectable labels such as biotin, fluorophores, chromophores, spin resonance probes or radiolabels. Fluorophores Examples include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminol). A fluorophore can be environmentally sensitive such that its fluorescence changes when it is in proximity to one or more residues in a modified protein that undergoes a structural change upon binding to a substrate (e.g., dansyl probe). Exemplary radiolabels include small molecules containing one or more atoms with low-sensitivity nuclei (13C, 15N, 2H, 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, 111In, etc.).

[0255] The diagnostic moiety includes a detectable moiety suitable for revealing the presence of a disease or disorder. Typically, the diagnostic moiety enables determination of the presence, absence, or level of a molecule associated with the disease or disorder, e.g., a target peptide, protein(s). Such a diagnosis is also suitable for predicting and / or diagnosing the disease or disorder and its progression.

[0256] Examples of therapeutic moieties include anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-infective agents, or generally, therapeutic drugs. The functional moiety can also have one or more of the above functions. Exemplary therapeutic moieties include antibiotics, a second anti-PD-1 antibody or an antibody to another antigen such as a tumor-specific antigen, an autoimmune tissue antigen, a virus-infected cell antigen, an Fc receptor, a T cell receptor or a T cell co-inhibitor or an immunotoxin or any other therapeutic moiety useful for treating a disease or condition including cancer, an autoimmune disease or a chronic viral infection. Exemplary therapeutic moieties can also include a cytotoxin, a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a toxin, a peptide or a therapeutic drug at any position along the molecule as long as it can bind to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In certain embodiments, the antibody is conjugated to a drug specific for tumor cells or virus-infected cells.

[0257] For example, a salvage receptor binding epitope, such as that described in U.S. Patent No. 5,739,277, can be attached to an antibody or an antigen-binding fragment thereof (particularly, an antibody fragment) to increase the half-life of the antibodies or antigen-binding fragments described herein. The term "salvage receptor binding epitope" may refer to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is involved in increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie et al., 18 Ann. Rev. Immunol. 739 (2000).

[0258] Nucleic acid Also provided herein are nucleic acids, vectors, host cells, and expression systems encoding the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein. The term "nucleic acid" as used herein refers to a polymeric form of nucleotides of either ribonucleotides or deoxyribonucleotides of any length, and includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The nucleic acids encoding the anti-PD-1 antibodies and antigen-binding fragments thereof and fusion proteins disclosed herein can be, for example, recombinant chimeric nucleic acid molecules containing any one or a combination of DNA, cDNA, RNA, synthetically produced DNA or RNA, or polynucleotides thereof.

[0259] The term "vector" refers to a vehicle containing a nucleic acid molecule capable of transporting the nucleic acid molecule into a cell. "Vectors" include, but are not limited to, viral vectors, plasmids, R An NA vector or a linear or circular DNA or RNA molecule is included, which may consist of chromosomal, extrachromosomal, semi-synthetic or synthetic nucleic acids. In some embodiments, the vectors used are those that enable self-replication (episomal vectors) and / or expression of the nucleic acids to which they are ligated (expression vectors). Several suitable vectors are known to those skilled in the art and are commercially available.

[0260] Antibody and fusion protein preparations and expression systems The anti-PD-1 antibodies, antigen-binding fragments or fusion proteins disclosed herein are usually produced by recombinant expression. Optionally, nucleic acids encoding the light and heavy chain variable regions linked to the constant regions can be inserted into the same expression vector. Alternatively, optionally, nucleic acids encoding the light and heavy chain variable regions linked to the constant regions are inserted into different expression vectors. The expression vector may further comprise one or more expression control sequences including, but not limited to, a promoter (e.g., a homologous or heterologous promoter), a signal sequence, an enhancer element and a transcription termination sequence. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. Usually, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of cross-reactive antibodies after the vector is incorporated into the appropriate host.

[0261] Generally, the expression vector contains a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to enable detection of cells transformed with the desired DNA sequence.

[0262] The host used to express the anti-PD-1 antibody, antigen-binding fragment or fusion protein disclosed herein can be a prokaryotic or eukaryotic host. Examples of suitable hosts include bacteria or eukaryotic hosts containing yeast, insect, fungal, avian and mammalian cells, or host cells of mammalian, insect, avian or yeast origin, either in vivo or in situ. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine or feline origin, although any other mammalian cells can be used.

[0263] Examples of bacterial hosts that can be used to express the antibodies, antigen-binding fragments or fusion proteins disclosed herein include Escherichia coli, bacilli such as Bacillus subtilus, and other enterobacteriaceae such as species of the genus Salmonella, Serratia, and various species of the genus Pseudomonas.

[0264] Yeast can also be used as a host for expressing the antibodies, antigen-binding fragments or fusion proteins disclosed herein. Saccharomyces and Pichia are exemplary yeast hosts, and suitable vectors have, as required, expression control sequences (e.g., promoters), origins of replication, termination sequences, etc. Common promoters include those for 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, cytochrome C, and enzymes involved in the utilization of methanol, maltose, and galactose.

[0265] Mammalian cells in culture can also be used as host cells that express the antibodies, antigen-binding fragments or fusion proteins disclosed herein. Examples of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) well-known in the art include CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells and hybridomas. Expression vectors for these cells include an origin of replication, expression control sequences such as promoters, enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites and / or transcription terminator sequences. Examples of expression control sequences include SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc.

[0266] The anti-PD-1 antibodies, antigen-binding fragments and fusion proteins disclosed herein can be expressed using a single expression construct or vector or multiple expression constructs or vectors (e.g., two or three expression constructs). When the heavy and light chains of the antibody are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of the intact immunoglobulin. Once expressed, all antibodies, their dimers, individual light and heavy chains or other immunoglobulin forms disclosed herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, etc. (generally, see Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). For pharmaceutical use, substantially pure immunoglobulins with at least about 90-95% homogeneity are preferred, and 98-99% or higher homogeneity is most preferred.

[0267] The disclosed anti-PD-1 antibodies, antigen-binding fragments and fusion proteins can be made by any method known in the art. General techniques for generating human or mouse antibodies or fusion molecules are known in the art.

[0268] Method for modulating PD-1 activity In one aspect, the present disclosure provides a method of using an anti-PD-1 antibody, antigen-binding fragment thereof, and fusion protein described herein to reduce the interaction between PD-1 and PD-ligands including, but not limited to, PD-L1 and PD-L2. In some embodiments, the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein disrupts the interaction between PD-1 monomers.

[0269] The anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein are useful for reducing immunosuppression, e.g., T cell tolerance. "Reducing" means the ability to cause an overall reduction of about 20% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more compared to an untreated control. Immunosuppression is mediated by the interaction of immunosuppressive receptors expressed on the surface of immune cells with their ligands. Methods for measuring T cell activity are known in the art. By way of non-limiting example, T cell tolerance can be induced by contacting T cells with recall antigen, anti-CD3 in the absence of costimulation and / or ionomycin. For example, the levels of IL-27, LDH-A, RAB10, and / or ZAP70 (both intracellular and secreted) can be monitored to determine, e.g., the extent of T cell anergy (using the levels of IL-2, interferon-γ, and TNF that correlate with increased T cell tolerance).

[0270] The anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein are further useful for enhancing T cell expansion, activation, and proliferation.

[0271] Treatment method PD-1 plays an important role in the immune system in the regulation of autoimmunity, tumor immunity, and infection immunity. PD-1-mediated T cell inhibition is an important mechanism for preventing autoimmunity. Furthermore, cancer and chronic infectious diseases can breach this regulatory mechanism and drive immunosuppression. Blocking PD-1 using antagonists, including monoclonal antibodies, has been studied in the treatment of cancer and chronic viral infections. The ability of PD-1 to inhibit T cell activation is exploited by chronic viral infections and tumors to evade the immune response. In chronic viral infections, PD-1 is highly expressed on virus-specific T cells, and these T cells become "exhausted" and lose effector function and proliferative capacity.

[0272] PD-1-deficient animal models develop various autoimmune phenotypes, including autoimmune cardiomyopathy, rheumatoid arthritis, and graft-versus-host disease. Specifically, they have high levels of IgG2b and IgA and develop mild lupus-like autoimmunity and dilated cardiomyopathy. Considering the role of PD-1 in modulating the immune response, the therapeutic agents disclosed herein that antagonize PD-1 signaling can be administered to treat diseases involving PD-1-mediated immunosuppression.

[0273] Immune regulation is a useful therapeutic approach for treating various diseases and disorders. One approach for immune regulation is to intervene at one or more immune checkpoints by regulators of immune activation that play important roles in maintaining immune homeostasis and preventing autoimmunity. Depending on the disease or disorder, it may be desirable to upregulate or downregulate the immune response. Tumor cells presenting non-self antigens can avoid immune attack by secreting cytokines or ligands that activate immune checkpoints. Thus, in cancer therapy, it is generally desirable to upregulate the immune response against tumor cells. In contrast, in the treatment of autoimmune diseases, it is generally desirable to downregulate the immune response in a particular tissue.

[0274] In one aspect, the present disclosure provides anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins useful for treating a subject in need thereof.

[0275] In the methods described herein, a therapeutically effective amount of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein disclosed herein is administered to a mammal in need thereof. The term "mammal" as used herein includes, but is not limited to, humans, laboratory animals, companion pets, and livestock. Preferably, the mammal is a human. As used herein, "therapeutically effective amount" refers to the amount of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein that is effective to produce a desired therapeutic effect when administered to a mammal.

[0276] "Subject" means a mammal including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, sheep, or cat. An individual and a patient are also subjects herein.

[0277] As used herein, the terms "treating," "treated," "treatment," or "treat" refer to therapeutic treatment, where the purpose is to decelerate (reduce), or obtain a beneficial or desired clinical result, a non-desirable physiological state, disorder, or disease. For purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the degree of a state, disorder, or disease; stabilization of a state, disorder, or disease situation (i.e., not worsening); delay or deceleration in the development of the progression of a state, disorder, or disease; improvement in one or more symptoms of a state, disorder, or disease situation; and remission, whether partial or complete. Treatment includes eliciting a clinically significant response without accompanying excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival in the absence of receiving treatment. The terms "preventing," "prevention," etc. refer to acting prior to the onset of a distinct disease or disorder to prevent the disease or disorder from occurring, or to minimize the degree of the disease or disorder, or to decelerate the process of onset. Treatment includes eliciting a clinically significant response without accompanying excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival in the absence of receiving treatment.

[0278] In one aspect, the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be used to treat subjects suffering from autoimmune diseases including, but not limited to, alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory muscle disease, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus, erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis. In certain embodiments, the activating antibodies disclosed herein can be used to treat subjects suffering from autoimmune diseases.

[0279] In one aspect, the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be used to treat subjects suffering from chronic viral infections. In certain embodiments, the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be used to rescue exhausted T cells and / or reduce viral titers in a subject in need thereof. In certain embodiments, chronic viral infections can be treated by administering a therapeutically effective dose of the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein to a subject in need thereof. In some embodiments, the subject is suffering from a viral infection caused by lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus (HIV), or human papillomavirus (HPV), or hepatitis B / C virus (HBV / HCV), or simian immunodeficiency virus (SIV).

[0280] In another aspect, the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be used to treat subjects suffering from primary or recurrent cancers including, but not limited to, renal cell carcinoma, colorectal cancer, non-small cell lung cancer, brain cancer (e.g., glioblastoma multiforme), squamous cell carcinoma of the head and neck, gastric cancer, prostate cancer, ovarian cancer, kidney cancer, breast cancer, multiple myeloma, and melanoma.

[0281] In certain embodiments, the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be administered in a therapeutically effective amount to a subject suffering from cancer or a viral infection.

[0282] One or more of the anti-PD-1 antibodies, antigen-binding fragments or fusion proteins disclosed herein can be administered to alleviate, prevent or reduce the severity of one or more of the symptoms or conditions of a disease or disorder. In certain embodiments, the anti-PD-1 antibodies, antigen-binding fragments and fusion proteins disclosed herein can be prophylactically administered to a subject in need thereof to reduce the risk of developing a chronic viral infection or an autoimmune disease. The anti-PD-1 antibodies, antigen-binding fragments and fusion proteins disclosed herein can be used as adjuvant therapy with any other agent or any other therapy known to those of skill in the art useful for treating cancer, autoimmune diseases or viral infections.

[0283] Combination therapy The anti-PD-1 antibodies, antigen-binding fragments and fusion proteins disclosed herein can advantageously be combined with additional therapeutic agents. Such additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitors, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, immunosuppressive agents and anti-infective agents. Administration of the anti-PD-1 antibody or its antigen-binding fragment or fusion protein and the additional therapeutic agent can be simultaneous, sequential or intermittent. Administration of the anti-PD-1 antibody or its antigen-binding fragment or fusion protein and the additional therapeutic agent can be separate or as a mixture. Further, the methods of treatment provided herein can be related to treatment in combination with one or more therapies including, but not limited to, the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy and radiation therapy.

[0284] The anti-PD-1 antibodies, antigen-binding fragments, and fusion proteins of the present disclosure can be used in synergistic combination with one or more anti-cancer drugs or therapies, including but not limited to, renal cell carcinoma, colorectal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, non-small cell lung cancer, colon cancer, ovarian cancer, adenocarcinoma, prostate cancer, glioma, and melanoma. Examples of such agents include, but are not limited to, a second antibody against PD-1 (e.g., nivolumab), an LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, another T cell co-inhibitor or an antagonist of a ligand (e.g., CD-28, 2B4, LY108, LAIR1. Antibodies against ICOS, CD160 or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., "VEGF-Trap" such as aflibercept or other VEGF-inhibitory fusion proteins or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib or pazopanib)], Ang2 inhibitors (e.g., nesvacumab), transforming growth factor β (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), agonists for co-stimulatory receptors (e.g., agonists for glucocorticoid-induced TNFR-related proteins), antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), antiviral drugs (e.g., zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobimetinib, tenofovir, rilpivirine and corticosteroids), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1 and CA19-9), vaccines (e.g., Bacillus Calmette-Guerin, cancer vaccines), adjuvants for increasing antigen presentation (e.g., granulocyte macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibody, PSMAxCD3 bispecific antibody), cancer vaccines (e.g., MAGE3, MUC1, EGFRv3, ALVAC-CEA), cytotoxics, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel and vincristine), cyclophosphamide, radiotherapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21 and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4Included are ADCs (such as anti-DS6-DM4 ADCs), anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, or any palliative care, radiotherapy, and / or antibodies against Fc receptors on immune cells for the treatment of cancer and / or autoimmune diseases.

[0285] Route of administration A therapeutic composition comprising any of the anti-PD-1 antibodies, antigen-binding fragments, or fusion proteins described herein can be administered to a subject in need thereof in any convenient manner including, but not limited to, by injection, infusion, implantation, or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intra-articularly, intramedullary, intramuscularly, intracranially, by intravenous or lymphatic injection, or intraperitoneally to a subject in need thereof. In one embodiment, the cell compositions of the disclosure are preferably administered by intravenous injection.

[0286] In certain embodiments, the anti-PD-1 antibody, antigen-binding fragment, or fusion protein is administered to a mammal by intravenous infusion, i.e., introduction of the anti-PD-1 antibody, antigen-binding fragment, or fusion protein into the vein of the mammal over a particular period of time. In certain embodiments, the period is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0287] Dosing regimen The method according to this aspect of the present disclosure includes sequentially administering multiple doses of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). The present disclosure includes a method of sequentially administering a single initial dose of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure to a patient, followed by one or more second doses of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure, and optionally, subsequently, one or more third doses of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure. The anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure can be administered at a dose between 0.01 mg / kg and 100 mg / kg.

[0288] In certain embodiments, the dose of the compound or composition is administered to the subject daily, every other day, every few days, every third day, once a week, twice a week, three times a week, once every two weeks, or once a month. In other embodiments, two, three, or four doses of the compound or composition are administered to the subject daily, every few days, every third day, once a week, once every two weeks, or once a month. In some embodiments, the dose of the compound or composition is administered for 2, 3, 5, 7, 14, 21, or 28 days. In certain embodiments, the dose of the compound or composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0289] Pharmaceutical composition In another aspect, there is provided a pharmaceutically acceptable composition comprising a therapeutically effective amount of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure formulated together with one or more pharmaceutically acceptable excipients.

[0290] The dosage of the active agent can vary depending on reasons of use, the individual subject, and the mode of administration. The dosage can be adjusted based on the subject's body weight, age, and health, as well as the tolerance to the compound or composition.

[0291] The active agent and the excipient can be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions provided herein can be specially formulated, for example, in solid or liquid forms adapted for parenteral administration by subcutaneous, intratumoral, intramuscular, or intravenous injection, such as sterile solutions or suspensions.

[0292] A therapeutic composition comprising an anti-PD-1 antibody or an antigen-binding fragment thereof or a fusion protein thereof can be formulated with one or more pharmaceutically acceptable excipients, which can be liquid or solid fillers, diluents, carriers, manufacturing aids (such as lubricants, magnesium talc, calcium stearate, zinc stearate, or stearic acid), solvents or encapsulating materials involved in the transport or delivery of the therapeutic compound for administration to a subject, bulking agents, salts, surfactants, and / or preservatives, pharmaceutically acceptable materials, compositions, or media. Some examples of materials that can act as pharmaceutically acceptable excipients are as follows: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations.

[0293] Excipients as referred to in this specification are described as compounds added to increase the mass of a pharmaceutical composition and to contribute to the physical structure of the formulation in lyophilized form. Examples of excipients include, but are not limited to, suitable mannitol, glycine, polyethylene glycol, and sorbitol.

[0294] The therapeutic composition may optionally include a surfactant. The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce the formation of particles in the reconstituted formulation. Examples of suitable surfactants that may be used in accordance with the present disclosure include, but are not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; methyl cocoyl taurate sodium, or methyl oleyl taurate disodium; and polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., pluronic, PF68, etc.).

[0295] In the therapeutic compositions described herein, preservatives may optionally be used. Suitable preservatives for use in the formulations provided herein include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl-dimethyl ammonium chlorides where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include phenol, aromatic alcohols such as butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0296] The therapeutic compositions described herein may have variable concentrations of anti-PD-1 antibodies, antigen-binding fragments thereof, or fusion proteins. For example, the composition may contain an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein at a concentration of 10 mg / ml to 200 mg / ml, 25 mg / ml to 130 mg / ml, 50 mg / ml to 125 mg / ml, 75 mg / ml to 110 mg / ml, or 80 mg / ml to 100 mg / ml. The composition may also contain an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein at about 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml. In some embodiments, the therapeutic composition can be provided as a lyophilized composition for reconstitution prior to administration.

[0297] Diagnostic uses The anti-PD-1 antibodies, antigen-binding fragments thereof, or fusion proteins of the present disclosure can be used to detect and / or measure PD-1 in a sample, for example, for diagnostic purposes. The anti-PD-1 antibodies, antigen-binding fragments thereof, or fusion proteins disclosed herein can be used in an assay to detect a disease or disorder such as cancer, an autoimmune disease, or a chronic viral infection. Exemplary diagnostic assays for PD-1 may include, for example, contacting a sample obtained from a patient with an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure, wherein the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein is labeled with a detectable label or reporter molecule or used as a capture ligand for selectively isolating PD-1 from the patient sample, or alternatively, used in combination with a secondary antibody that is itself detectably labeled. Detectable labels or reporter molecules can be isotopes such as 3H, C, 32P, 35S, or 25l; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; or enzymes such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure PD-1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0298] The kits of the present disclosure can include any combination of the agents, compositions, components, reagents, delivery devices or mechanisms, or other entities provided herein. For example, the kits of the present disclosure can include one or more anti-PD-1 antibodies or antigen-binding fragments thereof or fusion proteins thereof, and one or more of a carrier composition, a delivery device, and a combination therapy agent. The kit can further include a device that facilitates delivery, such as a syringe for injection, or a tool that facilitates delivery of a therapeutic composition to a subject in need thereof. Any of the kits provided herein can be included in a container, pack, or dispenser, together with instructions for use for administration.

[0299] All other cited patents and applications are hereby incorporated by reference in their entirety. Further, if the definition or use of a term in the references incorporated by reference herein is inconsistent with or contrary to the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the references shall not apply.

[0300] It should be understood that the present disclosure is not limited to the specific molecules, compositions, methodologies or protocols described, as these may change. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Further, it should be understood that the present disclosure herein includes all possible combinations of such specific configurations. For example, if a particular configuration is disclosed in the context of a particular aspect or embodiment of the present disclosure herein or of a particular claim, it can be combined with, and / or in the context of, other particular aspects and embodiments of the present disclosure, and furthermore, in the context of the present disclosure generally, that configuration can be used to the extent possible.

[0301] If a method involving two or more defined steps is mentioned herein, the defined steps can be performed in the order listed, or in any order, or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps that are performed before any of the defined steps, between two of the defined steps, or after all of the defined steps (except where the context excludes those possibilities).

[0302] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are presented. The following examples should not be read as limiting or defining the full scope of the present disclosure.

Examples

[0303] Identification of anti-PD-1 antibodies from the Distributed Bio SuperHuman 2.0 library hPD-1-Fc (human IgV domain fused to the N-terminus of human IgG1 Fc; exists as a dimer in solution) or hPD-1-his (human IgV domain of PD-1 connected to the N-terminus of a 6-histidine peptide; exists as a monomer in solution) was immobilized on immunotubes coated with anti-his antibody or anti-hFc antibody. Biotinylated hPD-1-his was captured by magnetic beads conjugated with streptavidin. The phage library was blocked with 3% PBS milk and added to the antigen-coated tubes or mixed with the antigen-coated beads and incubated at room temperature for 0.5 - 1 hour to allow the phages to bind to the immobilized antigen. Unbound phages were washed away, while the bound phages were eluted using triethylamine (pH > 11). The neutralized phages were used to infect log-phase Escherichia coli, which was grown overnight at 30°C. The recovered bacteria were used to amplify the phages for the next round of panning.

[0304] The heavy and light chain variable domains of the selected scFv antibodies isolated from the phage library were amplified by PCR and inserted into a mammalian expression vector containing a modified human IgG1 constant domain with effector function removed. Full-length IgG antibodies were expressed in HEK293 cells and purified by protein A. The purified anti-19B6, 19B10, 23A8, 23H9, 24H9 and 23A11 (see SEQ ID NOs: 1 - 48, Table 25) were characterized by binding (soluble and cell-expressed PD-1) and blocking assays, Biacore kinetic analysis and cytokine secretion in human PBMCs stimulated by SEB.

[0305] To identify higher affinity antibodies, two light chain shuffling libraries were constructed by combining the heavy chains of the lead antibodies 23H9 or 24H9, respectively, with the light chain mixture from R1 panning. Panning under more restrictive conditions identified higher affinity and / or more stable variants. These mature antibodies were converted to IgG, expressed, purified, and characterized again. Higher affinity clones were expressed in CHO cells and purified by Protein A and SE-HPLC to obtain higher quality for further studies. Antibodies 31B1, 33C4, 33G8, 34C1 (see SEQ ID NOs: 49-65, Table 25) were obtained by light chain shuffling using the heavy chain of 23H9. Antibodies 32A11, 32D11, 32D2, 32G6, 38A10, 38A11, 38A4, 38A6, 38A8, 38B1, 38B10, 38B2, 38C11, 38C6, 38G11, 38G9, 38H3, 39A3, 39B11, 39B3, 39B6, 39F11, 39G5, 39G8, 39H11, 39H2 and 39H7 (SEQ ID NOs: 66-138, see Table 25 ) were obtained.

[0306] Figure 1 represents the sequence alignment of exemplary antibodies generated herein. Table 25 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-PD-1 antibodies according to the Kabat CDR definition. The biological properties of the exemplary antibodies generated according to the method of this example are described in detail in the examples shown below.

Example

[0307] Antibodies isolated from the phage display library bind to hPD-1 The ability of anti-hPD1 antibodies 19B6, 19B10, 23A8, 23H9, 24H9, 23A11 (isolated from HEK293 cells) to bind to hPD1 was evaluated using a dose-response binding ELISA using the antibodies shown as primary antibodies. For this, high-binding (Immulon 2HB) plates were directly coated with human PD-1-Fc (R&D, catalog number 1086-PD-050). Biotinylated hPD-1-his (Sino Biologicals, catalog number 10377-H08H) was captured by streptavidin (Jackson 016-000-114) or anti-his antibody (R&D, catalog number MAB050-100) coated on the plate 1 day before. Serial dilutions of antibodies 19B6, 19B10, 23A8, 23H9, 24H9 and 23A11 were added to the antigen-coated plates and incubated at room temperature for 1-2 hours. After washing the plates 3 times with PBST (0.05%), anti-hFab-HRP antibody (Jackson 109-036-097) diluted 1 / 5000 - 1 / 10,000 was added to the plates and incubated at room temperature for 1 hour. Color was developed by adding a 1:1 dilution of substrate A and B for TMB (Fisher, catalog number 5120-0050), followed by neutralization with 1N H2SO4. OD450 was read using a TECAN plate reader (Table 1). Using GraphPad Prism8, the mean ± S.D. of the OD450 readings was plotted against the log antibody concentration. The EC50 value was calculated by plotting log(agonist) vs. response (3-parameter) or log(agonist) vs. slope of response with change (4-parameter) (Figure 2). The data points in Figure 2 represent the mean ± S.D. of duplicate quantifications. When bound to human PD-1-Fc, all 6 antibodies showed similar EC 50 and OD 450 (10 nM) values, but when bound to PD-1-his, different EC 50 and OD 450(10 nM) values were shown (Table 1). Antibodies 23A8, 23H9 and 24H9 showed excellent binding to hPD-1-his compared to other antibodies (Figure 2). Three lead antibodies - 23A8, 23H9 and 24H9 were selected based on their binding to PD-1-his (Table 1 and Figure 2).

[0308] [Table 1] [Examples]

[0309] Antibodies isolated from the phage display library block the hPD-1 / hPD-L1 interaction. To examine the ability of anti-hPD1 antibodies 19B6, 19B10, 23A8, 23H9, 24H9, 23A11 (isolated from HEK293 cells) to block the hPD-1 / hPD-L1 interaction, high-binding (Immulon 2HB) plates were coated with human PD-L1-Fc (R&D, catalog number 156-B7-100) or PD-L2-Fc (R&D, catalog number 1224_PL), respectively, and blocked with 3% PBS milk. Serial dilutions of antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 were mixed with biotinylated hPD-1-Fc at 0.5 μg / ml (final concentration) and incubated at room temperature for 1 hour. The mixture was transferred to plates coated with PD-L1-Fc or PD-L2-Fc and incubated at room temperature for an additional 1-2 hours. After washing the plates three times with PBST (0.05%), streptavidin-HRP (Thermofisher, catalog number 21140) diluted 1 / 5000-1 / 10,000 was added to the plates and incubated at room temperature for approximately 1 hour. Color was developed by adding a 1:1 dilution of substrate A and B for TMB, followed by neutralization with 1N H2SO4. OD450 was read using a TECAN plate reader (Table 2). Using GraphPad Prism8, the mean ± S.D. of the OD readings was plotted against the log antibody concentration. EC50 values were calculated by analyzing log(agonist) vs. response (3-parameter) or log(agonist) vs. slope of response with change (4-parameter). Data points for duplicate quantifications are mean ± S.D. (Figure 3). All six antibodies were found to block hPD-1-Fc binding to human PD-L1 and had different IC50 values (Table 2 and Figure 3). All six antibodies had very similar affinities for hPD-Fc. However, antibodies 23A8, 23H9, and 24H9 showed excellent blocking activity against hPD-1-his compared to the other antibodies (Table 2 and Figure 3).The three lead clones 23A8, 23H9 and 24H9 showed >93% inhibition and ≥70% inhibition in the presence of 30 and 3.3 nM antibody, respectively (Table 2 and Figure 3).

[0310]

Table 2

Example

[0311] Antibodies isolated from a phage display library bind to cells expressing human PD-1 Binding of anti-PD-1 antibodies to HEK293 and Jurkat cells overexpressing human PD-1: Cells were cultured in IMDM medium containing glutamine supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were first incubated with the indicated anti-PD-1 antibodies, followed by incubation with PE-anti-human IgG1-Fc (goat anti-human IgG conjugated with R-phycoerythrin: Jackson ImmunoResearch catalog number 109-116-098). Antibodies binding to hPD-1 were determined using a Guava EasyCyte™ HT sampling flow cytometer (EMD Mill ipore). The data points in Figure 4 represent the mean ± S.D. of duplicate quantifications. Antibodies 23A8, 23H9 and 19B10 showed the strongest binding to HEK293 cells, and antibodies 23A8, 24H9 and 19B10 showed the highest mean fluorescence intensity (MFI) (Table 4 and Figure 4).

[0312] Binding of anti-PD-1 antibodies to SEB-activated human PBMC: PBMC were isolated from LeukoPak (leukapheresis concentrate containing high concentrations of blood cells including monocytes, lymphocytes, platelets, plasma, and erythrocytes) using Histopaque-1077 (Sigma) according to the manufacturer's instructions. PBMC were cultured at 2×10 6 cells / mL in 12-well plates containing IMDM (Gibco, Life Technologies) supplemented with 10% FBS (GE Healthcare HyClone) and activated with 1 μg / mL of SEB (Staphylococcal enterotoxin B, Sigma) for 3 - 4 days. The activated PBMC were first incubated with the indicated anti-PD-1 antibodies, followed by incubation with PE-anti-human IgG1-Fc (goat anti-human IgG conjugated with R-phycoerythrin: Jackson ImmunoResearch catalog number 109-116-098) and either anti-human CD4-APC (eBioscience clone RPA-T4 catalog number 17-0049-42) or anti-human CD8-APC (eBioscience clone RPA-T8 catalog number 17-0088-42). Antibodies that bind to PD-1 expressed on the cells were evaluated using a Guava EasyCyte™ HT sampling flow cytometer (EMD Millipore). Data points were generated by calculating the mean ± S.D. of duplicate quantifications (Table 3 and Figure 4). Antibodies 19B6, 23A8, 23H9, and 24H9 showed the highest MFI and lowest EC50 for binding to PBMC among the antibodies tested and were selected for further characterization (Table 3 and Figure 4).

[0313]

Table 3

Example

[0314] Antibodies isolated from the phage display library disrupt the PD-1 / PD-L1 interaction between cells To evaluate the ability of the selected anti-PD-1 antibodies to disrupt the binding of PD-1-expressing cells to PD-L1-expressing cells, the Promega PD-1 / PD-L1 blockade bioassay was used. This assay consists of two genetically engineered cell lines, PD-1 effector cells and PD-L1 aAPC / CHO-K1 cells. When co-cultured, the PD-1 / PD-L1 interaction inhibits TCR-mediated luminescence. PD-1 / PD-L When the 1 interaction is disrupted, TCR activation induces luminescence (by activation of the NFAT pathway), which can be detected by the addition of Bio-Glo™ reagent and quantification using a luminometer.

[0315] The experiments were performed according to the manufacturer's instructions. Briefly, PD-L1 aAPC / CHO-K1 cells were plated and incubated for 16 - 20 hours, after which increasing concentrations of anti-PD-1 antibody and PD-1 effector cells were added. After incubation at 37 °C for 6 hours, Bio-Glo™ reagent was added and luminescence (recorded as relative light units, RLU) was measured using a TECAN plate reader. The fold induction was calculated as RLU (induced - background) / RLU (antibody-free control - background). Data points are represented as the mean ± S.D. of duplicate quantifications (Figure 5). Antibodies 19B6, 23A8, 23H9 and 24H9 blocked the hPD-1 / hPD-L1 interaction and had different EC50s and fold inductions (Table 4 and Figure 5).

[0316]

Table 4

[0317] Antibodies 23H9 and 24H9 were selected for light chain shuffling, and the heavy chains of 23H9 or 24H9 were paired with a light chain library, respectively. Antibodies 31B1, 33C4, 33G8, 34C1 (see SEQ ID NOs: 49-65, Table 25) were obtained by light chain shuffling using the heavy chain of 23H9. Antibodies 32A11, 32D11, 32D2, 32G6, 38A10, 38A11, 38A4, 38A6, 38A8, 38B1, 38B10, 38B2, 38C11, 38C6, 38G11, 38G9, 38H3, 39A3, 39B11, 39B3, 39B6, 39F11, 39G5, 39G8, 39H11, 39H2 and 39H7 (see SEQ ID NOs: 66-138, Table 25) were obtained by light chain shuffling using the heavy chain of 24H9.

Example

[0318] Anti-hPD-1 antibody 23H9 and its derivatives bind to hPD-1 and block the hPD-1 / hPD-L1 interaction The ability of anti-hPD1 antibodies 23H9 and its derivatives 31B1, 33C4, 33G8 and 34C1 to bind to hPD1 and block the hPD-1 / hPD-L1 interaction was determined using the protocols described in Examples 2 and 3, respectively. The parental antibody 23H9 and its derivatives showed no significant difference in binding to biotin-hPD-1-his captured by hPD-1-Fc and streptavidin (Table 5 and Figure 6A). However, all derivatives were more potent than their parent 23H9 in binding to hPD-1-his captured by anti-his and in blocking the interaction of hPD-1-F / hPD-L1-Fc (Table 5 and Figure 6B). Two clones (31B1 and 33C4) were selected for further characterization (Table 5).

[0319]

Table 5

Example

[0320] The anti-hPD-1 antibody 24H9 and its derivatives bind to hPD-1 and block the hPD-1 / hPD-L1 interaction. The ability of the anti-hPD1 antibodies 24H9 and its derivatives 32A11, 32D2, 32D11, 38A6, 38A10, 38A11, 38B2, 38C11, 39B3, 39B11, 39G8 and 38H11 to bind to hPD1 and block the hPD-1 / hPD-L1 interaction was determined using the protocols described in Examples 2 and 3, respectively. The parental antibody 24H9 and its derivatives showed no significant difference in binding to hPD-1-Fc and hPD-1-his captured by streptavidin (Figure 7). However, all derivatives were found to be more potent than the parental 24H9 in binding to hPD-1-his captured by anti-his and in blocking the interaction of hPD-1-Fc-hPD-L1-Fc (Figure 7).

[0321] Two clones (38A6 and 38B2) were selected for further characterization (Table 6).

[0322] [Table 6] [Examples]

[0323] The binding profiles of the 23H9 and 24H9 derivatives indicate that these antibodies bind to different binding epitopes. A dose-response binding assay using 23H9, 31B1, 33C4, 24H9, 38A6 and 38B2 was performed according to the protocol disclosed in Example 4. The results are shown in Figure 8. The 23H9 and 24H9 derivatives were found to be more potent than their parental antibodies in binding to PD-1-expressing cells (Table 7). Furthermore, the 23H9 and 24H9 derivatives showed different binding profiles, indicating that they bind to different binding epitopes (Figure 8).

[0324] [Table 7]

Example

[0325] Antibodies 23H9, 24H9 and their derivatives disrupt the PD-1 / PD-L1 interaction between cells A PD-1 blocking bioassay was performed using antibodies 23H9, 31B1, 33C4, 24H9, 38A6 and 38B2 according to the protocol outlined in Example 5. All derivatives were found to be more potent than their parental antibodies 23H9 and 24H9 in blocking the hPD-1-hPD-L1 interaction (Figure 9). 38B2 and 31B1 were found to exhibit the lowest EC50 and the highest induction fold among the six antibodies tested (Table 8 and Figure 9).

[0326]

Table 8

Example

[0327] Effect of anti-PD-1 antibodies on cytokine production in SEB-activated PBMC The effect of anti-PD-1 antibodies on cytokine production using 38A6, 38B2, 31B1, and 33C4 in SEB-activated PBMCs derived from donor FA was evaluated by measuring the amount of cytokines released by PBMCs in culture medium. PBMCs were isolated from LeukoPak (leukapheresis concentrate containing high concentrations of blood cells including monocytes, lymphocytes, platelets, plasma, and red blood cells) using Histopaque-1077 (Sigma) according to the manufacturer's instructions. PBMCs were cultured at 1 × 105 cells per well in 96-well plates containing IMDM (Gibco, Life Technologies) supplemented with 10% FBS (GE Healthcare HyClone). Cells were activated with 0.01 μg / mL of SEB (Staphylococcal enterotoxin B, Sigma) for 2 days. Supernatants were collected for the measurement of IL-2 using a Duoset ELISA kit (R&D Systems) according to the manufacturer's instructions (Figure 10A).

[0328] The experiment was repeated using PBMCs derived from donor EA to obtain the EC50 values of antibodies 23H9, 31B1, 24 H9, and 38B2 (Figure 10B).

[0329] The IL-2 concentration increased in a dose-dependent manner in the presence of all antibodies (Figures 10A, 10B). The IL-2 concentration increased in the presence of 23H9 derivative 31B1 and 24H9 derivative 38B2, which were found to be more potent than their parental antibodies 23H9 and 24H9, respectively (Table 9 and Figure 10B).

[0330] [Table 9]

Example

[0331] Biacore analysis of selected 23H9 and 24H9 derivatives The binding kinetics of the antibodies to either hPD-1-Fc or hPD-1-his were measured by surface plasmon resonance (SPR) using a Biacore T200. The human PD-1-Fc or PD-1-his protein was immobilized on a Series S CM5 sensor chip at pH 5 using standard amine coupling chemistry. Antibodies 23H9 and its derivatives 31B1, 33C4, as well as 24H9 and its derivatives 38A6, 38B2, 32D11, 39G8, 38A10, and 38A11 were injected at a concentration in the range of 1.5 - 100 nM at 30 μl / min onto the immobilized surface using 1X HBSEP as the running buffer. The contact time (association phase) was 3 minutes. The dissociation time was 6 - 10 minutes. Regeneration was performed after each binding cycle using an injection of 20 mM HCl at a flow rate of 30 μl / min for 30 seconds. Sensorgrams were obtained at each concentration and the derived curves were fitted to a 1:1 Langmuir binding model using Biaevaluation software. Derivatives 31B1 and 33C4 were found to be more potent than the parental 23H9 in their binding to hPD-1-Fc (Table 10). All derivatives of 24H9 were found to be more potent than the parental antibody 24H9 in their binding to hPD-1-his and hPD-1-Fc.

[0332]

Table 10

Example

[0333] Measurement of the thermal stability of selected 23H9 and 24H9 derivatives by differential scanning calorimetry (DSC) Selected 23H9 and 24H9 derivatives, 31B1, 33C4, 38A6 and 38B2 were cloned and expressed by WuXi Biologics using WuXi's mammalian expression vectors and CHO knockout cell lines. All antibodies were purified using Protein A and SEC-HPLC. More than 2.5 mg of purified antibody was obtained from 20 ml of culture after Protein A and SEC-HPLC purification. The monomer percentage of all antibodies exceeded 96% with endotoxin levels lower than 1.0 EU / mg. Differential scanning calorimetry (DSC) was used to determine the stability of four antibodies. DSC analysis was performed using a Malven DSC system. Protein samples were first diluted to 1 mg / mL using formulation buffer prior to analysis. 400 μl of each formulation buffer was added to a 96-well plate as a reference, and 400 μL of the protein sample was added. Samples were heated from 10°C to 100°C at a heating rate of 90°C / hour in a capillary DSC system. DSC results (Tm onset and Tm values) were analyzed using the supplier's software. The minimum heat transition midpoints (Tm1) of 31B1, 33C4, 38A6 and 38B2 were 70.5, 74.0, 71.0 and 71.9 °C, respectively (Figure 11A). The monomer percentage of all antibodies exceeded 96% with endotoxin levels lower than 1.0 EU / mg (Table 11). 33C4 and 38B2 were found to be the most thermally stable among all the antibodies tested (Table 11).

[0334]

Table 11

Example

[0335] The selected 23H9 and 24H9 derivatives bind to hPD-1 and block hPD-1 / hPD-L1 and hPD-1 / hPD-L2 interactions Using the protocols outlined in Examples 2 and 3 respectively, binding and blocking ELISAs were performed using antibodies 31B1, 33C4, 38A6, and 38B2 isolated from transient CHOk cells. By comparison of the antibodies using binding ELISA, no significant differences were shown between the derivatives in their binding to hPD-1-Fc, biotinylated hPD-1his captured by streptavidin, and hPD-1-his captured by anti-his antibody directly bound to the plate (Figures 12A - C). However, the EC50s of 38B2 and 38A6 (both 24H9 derivatives) were lower than those of 31B2 and 33C4 (both 23H9 derivatives) and were superior to both controls (Table 12 and Figures 12A - C).

[0336] By comparison of the antibodies using blocking ELISA, no significant differences were shown between the derivatives in the blocking of hPD-1 / hPD-1 and hPD-1 / hPD-L2 interactions (Figures 12D - E). However, similar to the binding ELISA, antibodies 38A6 and 38B2 (both 24H9 derivatives) were found to be more potent than 31B2 and 33C4 (both 23H9 derivatives), particularly in inhibiting hPD-L2 binding to hPD-1 (Table 13 and Figures 12D - E).

[0337] [Table 12]

[0338] [Table 13] [Examples]

[0339] Dose - response binding assays using 33B1, 33C4, 38A6, and 38B2 Using the protocol outlined in Example 4, dose - response binding assays were performed using antibodies 33B1, 33C4 (both 23H9 derivatives), 38A6, and 38B2 (both 24H9 derivatives).

[0340] The dose-response binding assay showed no difference between two derivatives derived from the same parental antibody in the binding of the antibody to hPD-1 transfected HEK-293 and Jurkat cells and SEB-activated PBMC (Table 14 and Figure 13). However, derivatives derived from different parental antibodies showed differences in binding to different cell lines, suggesting that the 23H9 derivative and the 24H9 derivative have different binding epitopes (Table 14 and Figure 13).

[0341] [Table 14] [Example]

[0342] The selected 23H9 and 24H9 derivatives bind to hPD-1 with high affinity The binding of the selected antibodies to hPD-1 his and hPD-1 Fc was determined by SPR. Biacore analysis of 31B1, 33C4, 38A6 and 38B2 was performed using the protocol described in Example 11. All four antibodies were found to have high affinity for both hPD-1-Fc and hPD-1-his (Table 15 and Figure 14). However, between the two derivatives of 23H9, 31B1 was found to be more potent than 33C4 in both bindings to PD-1-Fc and PD-1-his (Table 15). Between the two of 24H9, 38B2 was found to be slightly more potent than 38A6 in the binding to hPD-1-Fc and showed no significant difference in the binding to hPD-1-his. Both 31B1 and 38B2 were found to be superior to the two controls (Table 15).

[0343] [Table 15] [Example]

[0344] Antibodies 31B1 and 38B2 disrupt the cell-cell PD-1 / PD-L1 interaction To evaluate the ability of 31B1 and 38B2 to disrupt the binding of PD-1-expressing cells to PD-L1-expressing cells, the Promega PD-1 / PD-L1 blocking bioassay was performed using the protocol described in Example 5. No significant differences in EC50 and fold induction were observed between 31B1 and 38B2 (Figure 15). Both 31B1 and 38B2 were found to be superior to control B (nivolumab, sold under the trade name Opdivo) and comparable to control M (pembrolizumab, sold under the trade name Keytruda) (Table 16).

[0345]

Table 16

Example

[0346] Effect of 31B1 and 38B2 on cytokine production in SEB-activated PBMC The effect of 31B1 and 38B2 on cytokine production in SEB-activated PBMC was determined using the protocol outlined in Example 4. 38B2 was found to be more potent than 31B1 and two control antibodies tested (Table 17 and Figure 16).

[0347]

Table 17

Example

[0348] Isolation of antibodies cross-reactive with human and mouse from the Distributed Bio SuperHuman 2.0 Inc. library A PD-1 antibody that binds to both mouse and human PD-1 was isolated using the protocol outlined in Example 1, using mPD-1-Fc (mouse IgV domain fused to the N-terminus of human IgG1Fc; exists as a dimer in solution) or mPD-1-his (mouse IgV domain of PD-1 connected to the N-terminus of six histidines; exists as a monomer in solution).

[0349] Derivatives of the anti-mPD-1 antibody R3A9 (SEQ ID NOs: 139-146) by light chain shuffling using the VH chain of R3A9. R3A9 and its derivatives (see, for example, SEQ ID NOs: 147-167) were selected and further developed. The antibodies were cloned and expressed by WuXi Biologics using Wuxi mammalian expression vectors and CHO knockout cell lines. The antibodies were purified by Protein A and SEC-HPLC. More than 2.5 mg of purified antibody was obtained from 20 ml of culture after Protein A purification. The antibodies were further purified by SEC-HPLC.

[0350] The biological properties of the selected antibodies generated according to the method of this example are detailed in the examples shown below. Figures 1 and 25 provide the amino acid sequences of the heavy and light chain variable regions and CDRs of the selected anti-mPD-1 antibodies (Kabat numbering).

Example

[0351] The anti-mPD-1 antibody R3A9 and its derivatives bind to mPD-1 and block the mPD-1 / mPD-L1 interaction Using the protocol as outlined in Examples 2 and 3, dose-response binding and blocking ELISAs were performed using R3A9 and its derivatives. Binding of the antibodies to cells expressing mPD-1 was performed as described in Example 4. Blocking of the intercellular PD-1 / PD-L1 interaction was examined using the Promega bioassay described in Example 5. The results are shown in Figure 17.

[0352] The R3A9 derivative m3A7 was found to be more potent than the parental R3A9 antibody in both binding to mPD-1 expressed by soluble cells and blocking the mPD-1 / mPD-L1 interaction (Table 18).

[0353] [Table 18] [Examples]

[0354] The R3A9 derivatives m2C1, m2A1, m4D1, and m3A7 bind to human and mouse PD-1 Using the protocols outlined in Examples 2 and 3, respectively, dose-response binding and blocking ELISAs were performed for the antibodies. The experiments showed that all R3A9 derivatives bound strongly to both hPD-1 and mPD-1 (Figures 18A - C) and blocked the hPD-L1-hPD-1 and mPD-L1 / mPD-1 interactions (Figures 18D - E). Table 19 provides the EC50 / IC50 data for the antibodies tested.

[0355] [Table 19] [Examples]

[0356] R3A9 binds to cells expressing human or mouse PD-1 The binding of R3A9 and its R3A9 derivatives m2A1, m2C1, m4D1, m3A7 to PB MC cells was tested using the protocol outlined in Example 4. Additionally, Biacore analysis was performed with all the antibodies disclosed in this example using the protocol provided in Example 11.

[0357] Only three derivatives, m4d1, m2A1, and m3C1, were found to bind to both human and mouse PD-1 expressed in cells (Table 21 and Figure 19). Based on cell binding (Table 20) and Biacore analysis (Table 20), m4D1 was found to exhibit improved binding characteristics compared to the other two antibodies tested (Table 20 and Figure 19).

[0358]

Table 20

[0359]

Table 21

Example

[0360] Promega (h / m) PD-1 / (h / m) PD-L1 Blocking Bioassay Comparing m4D1 with 38B2 and m3A7 Using the protocol outlined in Example 4, a Promega blocking bioassay was performed to compare the efficacy of m4D1, m3A7, and 38B2 in disrupting the PD-1 / PD-L1 interaction between cells. The results are presented in Figure 20.

[0361] m4D1 was found to block both hPD-1 / PD-L1 and mPD-1 / mPD-L1 interactions. Furthermore, m4D1 was found to be as potent as m3A7 in the mPD-1 / mPD-L1 blocking assay and one-third as potent as 38B2 (Figure 20).

Example

[0362] Fusion Protein Containing Anti-mPD1- or Anti-hPD-1 Antibody, IL-15, and IL-15Rα Sushi Domain An anti-mPD-1 fusion antibody containing antibody m3A7 (also called m3A7 / IL-15) was engineered by adding the sushi domain of IL-15Rα and the IL-15 polypeptide to either the C-terminus or N-terminus of the antibody. The fusion of the IL-15Rα sushi domain, the hinge region portion, linker 1 and IL-15 is called "SD15". For an example of the SD15 domain, refer to SEQ ID NO: 174 in Table 25, the SD15 domain highlighted in bold.

[0363] In the first version of the fusion protein, the SD15 domain was fused to the C-terminus of only one IgG1 CH1-CH2-CH3 domain (designated "1C-m3A7 / IL-15").

[0364] In the second version, the SD15 domain was fused to the C-terminus of both IgG1 CH1-CH2-CH3 domains (designated "2C-m3A7 / IL-15").

[0365] In the third version of the fusion protein, the SD15 domain was linked to the N-terminus of only one VH domain via a second linker (designated "1N-m3A7 / IL-15").

[0366] In the fourth version of the fusion protein, the SD15 domain was linked to the N-terminus of both VH domains via a second linker (designated "2N-m3A7 / IL-15").

[0367] In the fourth construct described above, the light chain was that of a conventional antibody. To eliminate complement binding and fixation as well as Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC), the amino acid substitutions L234A, L235A, P329G (LALA-PG) were introduced into both CH2 domains.

[0368] Furthermore, for heterodimer fusions, mutations were introduced into the constant region of the heavy chain, and the amino acid substitutions were selected from one of the following groups: (1) CH3-1: T366Y ("knob"); CH3-2: Y407T ("hole"), (2) CH3-1: S354C, T366W ("knob"); CH3-2: Y349C, T366S, L368A, Y407V ("hole") and (3) CH3-1: T350V, L351Y, F405A, Y407V ("knob"); CH3-2: T350V, T366L, K392L, T394W ("hole").

[0369] The fusion antibodies were transiently expressed in CHOk1 cells. Monomers with a purity of at least 95% were obtained after purification using Protein A and SEC-HPLC. Four corresponding versions of the anti-hPD-1 / IL-15 fusion antibody containing antibody 38B2 (described herein as 38B2-hSD15 or 38B2 / IL-15) - 1C-38B2-hSD15, 2C-38B2-hSD15, 1N-38B2-hSD15, 2N_-38B2-hSD15 - were generated according to the same procedure. Three corresponding versions of 1N_38B2-SD15 with an amino acid substitution at position 65 of the IL-15 sequence, 65A_1N _38B2-hSD15, 65D_1N_38B2-hSd15 and 65S_1N_38B2-hSD15 were generated using the same procedure.

[0370] Four corresponding versions of the control fusion antibody (DP47 / IL-15), 1C-DP47 / IL-15, 2C-DP47 / IL-15, 1N-DP47 / IL-15, 2N-DP47 / IL-15 were generated according to the same procedure. DP47 was used as an untargeted control antibody. A schematic diagram showing the antibody orientation is shown in Figure 21.

Example

[0371] The mPD-1 / IL-15 fusion molecule binds to mouse PD-1 and blocks the interaction between mPD-1 and mPD-L1 The ability of the fusion proteins 1N-m3A7 / IL-15, 1C-m3A7 / IL-15, 2N-m3A7 / IL-15, and 2C-m3A7 / IL-15 to bind to mPD-1 and block the interaction between mPD-1 and mPD-L1 was evaluated according to the protocols described in Examples 2 and 3, respectively.

[0372] No significant differences in binding and blocking ability were observed among the four different molecules (Table 22 and Figure 22).

[0373]

Table 22

Example

[0374] Effect of mPD-1 / IL-15 fusion molecules on IL-2-dependent cell growth stimulation of mouse lymphocytes CTLL2 and C57BL6 spleen cells CTLL2 culture and proliferation assay: Mouse T lymphocyte cell line CTLL2 was cultured in IMDM medium supplemented with 10% heat-inactivated FBS and 10% IL-2 supplement (T cell culture supplement containing ConA, Corning). In this example, the following fusion proteins were tested: 1N-m3A7 / IL-15, 2N-m3A7 / IL-15, 1C-m3A7 / IL-15, and 2C-m3A7 / IL-15. 1N-DP47 / IL-15, 2N-DP47 / IL-15, 1C-DP47 / IL-15, 2C-DP47 / IL-15, and m3A7 were used as controls.

[0375] Serial dilutions of the antibody or fusion protein were added to 96-well plates, and then the cells were seeded at 2.5 × 104 cells per well in IMDM medium supplemented with 10% heat-inactivated FBS. The cells were incubated at 37°C for 3 - 5 days, and cell proliferation was detected using the CellTiter-Glo (registered trademark) luminescent cell viability assay kit (Promega).

[0376] Mouse spleen cell isolation and proliferation assay: The spleens of C57BL / 6 mice were homogenized in IMDM medium using a 70 μm cell strainer. 1× RBC lysis buffer (eBioscience) was added to the spleen cells, and the cells were allowed to stand for 3 minutes with occasional shaking, and then diluted with 5 volumes of PBS buffer. After washing with PBS buffer, immediately, a spleen cell cell suspension was prepared with complete IMDM medium supplemented with 10% heat-inactivated FBS for the proliferation assay. In this example, the following fusion proteins were tested: 1N-m3A7 / IL-15, 2N-m3A7 / IL-15, 1C-m3A7 / IL-15, and 2C-m3A7 / IL-15. 1N-DP47 / IL-15, 2N-DP47 / IL-15, 1C-DP47 / IL-15, and 2C-DP47 / IL-15 were used as controls.

[0377] Serial dilutions of the fusion proteins and controls were added to 96-well plates, and then the cells were seeded at 1×105 cells per well. The cells were cultured at 37 °C for 5 - 7 days and collected for flow cytometry analysis. The cells were washed and stained with the fixable viability dye Fluor780 (eBioscience) for 30 minutes at 4 °C. After washing, the cells were fixed and permeabilized using the Foxp3 transcription factor staining buffer set (eBioscience), and subsequently stained with CD3 (145-2C11)-PE, CD4 (RM4-5)-APC, CD8α-(53-6.7)-PECy7, and Ki67 (SolA15)-FITC from eBioscience. Proliferative CD4 (CD4 + Ki67 + ) or CD8 (CD8 + Ki67 + ) T cell populations were analyzed and defined using the Guava flow cytometry EasyCyte system.

[0378] All mPD-1 / IL-15 fusion constructs stimulated both IL-2-dependent mouse lymphocyte CTLL2 (Figure 23A) and C57BL6 spleen cell proliferation (Figure 23B). Analysis of the T cell population showed that only the percentage of the CD8+ T cell population increased, while CD4 +The percentage of the T cell population was shown to remain unaffected (Figure 23C). Among the four constructs tested, 1N-m3A7 / IL-15 showed the minimal stimulatory effect on IL-2-dependent mouse lymphocyte and spleen cell proliferation and T cell proliferation, while 2C-m3A7 / IL-15 showed the most stimulatory effect on cell proliferation and T cell proliferation.

Example

[0379] Study on the efficacy of mPD-1 / IL-15 fusion molecule in PD-1 / PD-L1 Lewis lung cancer model To determine whether the orientation or number of the IL-15 / IL-15Rα sushi domain in the fusion protein has any effect on the ability of the fusion molecule to reduce tumor growth, an in vivo efficacy study in an LL / 2 mouse lung cancer syngeneic model was conducted by Crown Bioscience Inc. (Taicang) using the m3A7 / IL-15 / IL-15Rα sushi fusion protein. In this example, constructs 1N-m3A7 / IL-15 and 1C-m3A7 / IL-15 were tested, and 1N-DP47 / IL-15 and 1C-DP47 / IL-15 were used as controls.

[0380] Mice of the C57BL / 6 strain, 8 - 10 weeks old and weighing 16.5 - 20.7 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd (Shanghai, China). LL / 2 tumor cells were maintained in vitro at 37 °C in the presence of 5% CO2 using DMEM medium supplemented with 10% fetal bovine serum. Cells were harvested in the logarithmic growth phase, counted, and then tumor-inoculated. The fusion proteins used in this example were produced by WuXi Biologics, showed a purity of at least 95%, and contained more than 95% monomer.

[0381] In the right posterior abdominal region of each mouse, 0.1 ml of LL / 2 tumor cells (3×10 5(number) were subcutaneously inoculated. The date of randomization and the first dosing day were designated as day 0. When the average tumor size reached approximately 100 mm 3 the randomization process was initiated. Mice were randomly assigned to different study groups. Randomization was performed using the multi-task method (StudyDirector™ software, version 3.1.399.19) randomization block design and was based on the "matched distribution" method.

[0382] The fusion protein was diluted according to the indicated dose, and 10 μl / kg of the fusion protein was intravenously injected into the mice once a week for 3 weeks. After randomization, the mice were weighed twice a week. Tumor volume was measured twice a week in two dimensions using calipers, and the volume was expressed in mm3 using the formula: "V = (L × W × W) / 2 (where V represents the tumor volume, L represents the length of the tumor (the longest tumor dimension), and W represents the width of the tumor (the longest tumor dimension perpendicular to L)). Dosing and tumor and body weight measurements were performed in a Laminar Flow Cabinet. Body weight and tumor volume were measured using StudyDirector™ software (version 3.1.399.19).

[0383] To determine the anti-tumor activity of the fusion protein, the tumor growth inhibition (TGI) percentage was measured and recorded daily using the following formula: TGI(%) = 100 × (1 - T / C) (where T and C represent the average tumor volume (or body weight) of the treatment group and the control group for the fusion protein and the control, respectively).

[0384] Among the fusion proteins tested, 1N-m3A7 / IL-15 showed the most inhibitory effect on tumor growth (Table 23 and Figure 24).

[0385] In summary, the data obtained from Examples 25 and 26 suggest that the N-terminal fusion antibody further reduces toxicity compared to the C-terminal fusion antibody due to lower stimulation of peripheral T cells. The data further show that the N-terminal fusion molecule is particularly effective in stimulating tumor-infiltrating lymphocytes (TILs) compared to peripheral T cells for cis-presentation; the reduction of IL-2Rβγ binding in the N-terminal fusion molecule allows PD-1 / IL-15 to selectively bind to TILs and thus reduce its toxicity. Finally, the data show that the 1N-fusion molecule is superior to the 2N-fusion molecule.

[0386]

Table 23

Example

[0387] The hPD-1 / IL-15 fusion protein retained its PD-1 / PD-L1 inhibitory function. The Promega blockade bioassay was performed as described in Example 5 using the fusion proteins 1C-38B2-hSD15, 2C-38B2-hSD15, 1N-38B2-hSD15, 2N-38B2-hSD15, 1N-65D-hSD15-38B2 and 1N-65S-hSD15-38B2. All fusion molecules retained the PD-1 / PD-L1 inhibitory function compared to 38B2 (Figure 26). The 1N-fusion provided the most PD-1 / PD-L1 inhibitory function.

Example

[0388] The hPD-1 / IL-15 fusion protein stimulates IL-15 activity. ExaM07e culture and proliferation assay: To determine the ability of the fusion protein to stimulate IL-15 in vitro, the human acute megakaryoblastic leukemia M07e cell line was cultured in IMDM medium supplemented with 15% heat-inactivated FBS and 20% conditioned medium of the cell line 5637.

[0389] Serial dilutions of the antibody or fusion protein were added to 96-well plates, and then cells were seeded at 2.5×10 4 per well in IMDM medium supplemented with 10% heat-inactivated FBS. Cells were incubated at 37 °C for 3 - 5 days, and cell proliferation was detected using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega).

[0390] All tested fusion protein constructs stimulated IL-15 activity, but the 1N fusion showed a reduced ability to stimulate compared to the 1C, 2C, or 2N constructs (Figure 27).

Example

[0391] Study on the efficacy of the hPD-L1 / SD15 fusion molecule in the CT26 syngeneic model To determine whether the orientation or number of IL-15 / IL-15Rα sushi domains has any effect on the ability of the fusion molecule to inhibit tumor growth, in vivo efficacy studies were performed in the CT26 syngeneic model using 1N-SD15-D7A8, 2N-SD15-D7A8, 1C-SD15-D7A8, and 2C-SD15-D7A8. CT26 syngeneic model mice were treated with the fusion protein and observed for tumor development / reduction. CT26 is a mouse colon cancer tumor cell line.

[0392] Tumor volume was measured twice a week in two dimensions using calipers, and the volume was expressed in mm 3 using the formula: "V = (L × W × W) / 2, where V represents the tumor volume, L represents the length of the tumor (the longest tumor dimension), and W represents the width of the tumor (the longest tumor dimension perpendicular to L). Drug administration and tumor and body weight measurements were performed in a Laminar Flow Cabinet. Body weight and tumor volume were measured using StudyDirector® software (version 3.1.399.19).

[0393] The results of the efficacy study using the PD-L1 / IL-15 molecule are shown in Figure 28.

Example

[0394] The presence of the IL-15 amino acid substitution in the 1N-fusion protein did not affect the PD-1 / PD-L1 inhibitory function of the fusion protein. The Promega blocking bioassay (Figures 26C, 29E, and 29F) and ELISA competition assay (Figures 29B and 29C) showed that the N65S or N65D amino acid substitution in the 1N-fusion protein containing IL-15 did not affect the ability of the fusion protein to effectively disrupt the PD-1 / PD-L1 or PD-1 / PD-L2 interaction.

[0395] Similarly, no significant difference was observed in the strong binding of the 1N-fusion protein containing wild-type IL-15 or IL-15 N65S to cells expressing soluble PD-1 or PD-1, respectively (Figures 29A and 29D).

Example

[0396] The 1N-fusion protein containing the IL-15 amino acid substitution showed weak IL-15 stimulation in vitro. The ability of the 1N-fusion protein to stimulate IL-15 activity in human and mouse cells was examined. In the presence of N65S-1N-IL-15 / 38B2, human acute megakaryoblastic leukemia cells (M07e, Figures 30A, 30B, and 30C), hPBMC (Figure 30D), IL-2 / IL-15-dependent mouse lymphocyte cell line (CTLL2, Figure 31A), and mouse spleen cells (Figure 31B) proliferated more slowly than in the presence of wild-type 1N-IL-15 / 38B2. The binding of N65S-1N- / 38B2 to HEK293 transfected with hIL-2Rβ decreased (Figure 30E).

[0397] Furthermore, both 1N-IL-15 / 38B2 and mut-1N-IL-15 / 38B2 showed CD4 in both mouse spleen (Figure 31C) and hPBMC (Figure 31D). +Rather than T cells, CD8 + induced a better dose response in the proliferation of T cells.

Example

[0398] The 1N-fusion protein containing the IL-15 amino acid substitution showed strong anti-tumor activity in vivo in an anti-PD-1 antibody-resistant mouse model. The in vivo efficacy of 1N-fusion proteins containing wild-type IL-15, IL-15 N65S, or IL-15 N65D, respectively, was evaluated in hPD-1 / hPD-L1 transgenic BALB / c mice bearing Keytruda (anti-PD-1 antibody)-resistant hPD-L1-CT26 tumors. When the tumor size reached approximately 100 mm 3 fusion antibody (once a week) and control (twice a week) were injected (intraperitoneally).

[0399] Both 1N-38B2 / IL-15 and N65S-1N-38B2 / IL-15 showed strong anti-tumor activity (Figure 32A). The anti-tumor efficacy of N65S-1N-38B2 / IL-15 was dose-dependent (Figure 32B). N65S-1N-38B2 / IL-15 at 12 mg / kg, QW × 3 weeks demonstrated tumor regression (Figures 32B and 32C). When dosed at 12 mg / kg with N65S-1N-38B2 / IL-15, no weight loss or death was observed, but significant weight loss and death were observed when dosed with 1N-38B2 / IL-15 at ≥1 mg / kg (Figures 32E and 32E). The non-targeted 1N-IL-15 fusion showed no anti-tumor efficacy at all. Significant weight loss (Figure 32D) and death (data not shown) were observed when the non-targeted 1N-IL-15 fusion was dosed at ≥1 mg / kg.

[0400] The 1N-fusion protein containing IL-15 N65S showed an improvement in efficacy compared to the 1N-fusion protein containing IL-15 N65D (Figures 32I and 32K).

[0401] In short, the fusion protein containing mutant IL-15 reduced the efficacy and stimulation associated with IL-15, resulting in reduced toxicity and an increased therapeutic window.

Example

[0402] The 1N-fusion protein containing IL-15 amino acid substitution showed strong anti-tumor activity in vivo in mice re-challenged with tumor cells. In hPD-1 / hPD-L1 transgenic mice, the in vivo efficacy of the 1N-fusion protein containing IL-15 N65S or IL-15 N65D, respectively, was evaluated. Sex was evaluated.

[0403] 0.5×10 6 Individual hPDL1 / CT26 were subcutaneously inoculated into the lower right abdomen of mice. When the tumor size reached about 100 mm 3 Upon reaching, mice were intraperitoneally injected with 12 mg / kg of N65S-1N-38B2 / IL-15 or N65D-1N-38B2 / IL-15 once a week and twice a week for 3 weeks, respectively. In 5 mice (out of 6), tumors completely disappeared at about 25 days for N65S-1N-38B2 / IL-15 treatment and at 40 days for N65D-1N-38B2 / IL-15 treatment.

[0404] 6 Individual hPDL1 / CT26 cells and 0.1×10 6 Individual CT26 cells were subcutaneously inoculated into the lower left abdomen and upper left abdomen, respectively. No tumor growth was observed up to day 135 for both cell lines, indicating that the fusion protein protected mice from re-challenge with tumor cells (Figure 33).

Example

[0405] The 1N-fusion protein containing IL-15 amino acid substitution showed improved anti-tumor activity in hPD1 / PDL1 mice with hPDL1-CT26 tumors compared to the combination of an untargeted fusion protein and an anti-PD-1 antibody. 0.5×10 6 0.5×10^6 hPDL1 / CT26 cells were subcutaneously inoculated into the right lower abdomen of mice. When the tumor size reached approximately 100 mm 3 , the mice were intraperitoneally injected with 38B2 at 10 mg / kg, N65S-1N-38B2 / IL-15 at 12 mg / kg, and a combination of 38B2 at 10 mg / kg and N65S-1N-DP47 / IL-15 at 12 mg / kg once a week for 3 weeks. For the combination treatment, the two test substances were sequentially injected at 1-hour intervals.

[0406] The ability to suppress tumor growth was compared for the following constructs: (1) N65S-1N-38B2 / IL-15 fusion protein; (2) non-targeted fusion N65S-1N-DP47 / IL-15; (3) combination of anti-PD-1 antibody 38B2 and non-targeted fusion (i.e., N65S-1N-DP47 / IL-15); and (4) anti-PD-1 antibody 38B2 alone.

[0407] Tumors grew significantly slower in the N65S-1N-38B2 / IL-15 group compared to the combination group (Figure 34A). In the N65S-1N-38B2 / IL-15 fusion group, 83% (5 / 6) of the mice had no tumors, while no tumor-free mice were observed in the combination group, and one tumor-free mouse was observed in the group with anti-PD-1 antibody 38B2 alone (Figure 34B). No weight loss was observed in the 65S-1N-IL15 / 38B2 fusion group, and approximately -5% weight loss was observed in the non-targeted fusion 65S-1N-IL15 / DP47 group (Figure 34C).

[0408] This data demonstrates the robust anti-tumor activity of the N65S-1N-38B2 / IL-15 fusion protein, highlighting its bifunctionality and its ability to bind to both PD1 and IL2Rβγ co-expressed on TILs.

Example

[0409] Additional administration of anti-PD-1 antibody does not lead to further increase in the anti-tumor activity of the 1N-fusion protein 0.5×106 Individual hPDL1 / CT26 cells were subcutaneously inoculated into the right lower abdomen of mice. When the tumor size reached approximately 100 mm 3 upon reaching, mice were intraperitoneally injected with 10 mg / kg of 38B2, 12 mg / kg of N65S-1N-38B2 / IL-15, and a combination of 10 mg / kg of 38B2 and 12 mg / kg of N65S-1N-38B2 / IL-15 once a week for 3 weeks. For the combination treatment, the two test substances were first mixed and then injected into the mice.

[0410] The ability to suppress tumor growth was compared for the following constructs: (1) N65S-1N-38B2 / IL-15 fusion protein; (2) combination of anti-PD-1 antibody 38B2 and N65S-1N-38B2 / IL-15 fusion protein; and (3) anti-PD-1 antibody 38B2 alone.

[0411] Tumors grew significantly slower in the N65S-1N-38B2 / IL-15 group compared to the combination group (Figure 35A). In the N65S-1N-38B2 / IL-15 group, 83% (5 / 6) of the mice had no tumors, while no tumor-free mice were observed in the combination group, and only one tumor-free mouse was observed with the antibody alone (Figure 35B). Body weights are shown in Figure 35C.

[0412] This data indicates that the 1N-fusion protein is more effective in reducing tumor growth than the combination of the 1N-fusion protein and the anti-PD-1 antibody.

Example

[0413] The N65S-1N-38B2 / IL-15 fusion protein showed a long serum half-life in hPD1 / PDL1 transgenic mice with hPDL1-CT26 tumors hPD1 / PDL1 transgenic Balb / c mice were subcutaneously inoculated in the right lower abdomen with hPDL1 / CT26 tumor cells (0.5×10 6 cells) in 0.1 mL of PBS. The animals were randomized, and the average tumor volume was approximately 83 mm3 Treatment was initiated when [the condition] reached [a certain state]. 38B2 at 5.1 mg / kg (0.35 μmol / kg) or 1 or 6 mg / kg (0.06 or 35 μmol / kg) of N65S-1N-38B2 / IL-15 was administered intraperitoneally (IP) or intravenously (IV) to mice. After dosing, blood was collected at 0.2 hours, 5 hours, 2 hours, 4 hours, 12 hours, 24 hours, 48 hours, 72 hours, 120 hours, and 168 hours after injection. Using purified serum, the antibody concentration was measured by a traditional ELISA that used human PD1 as the captured protein and biotin-IL15 and anti-human Fab separately to detect KD050 and 38B2 binding.

[0414] N65S-1N-38B2 / IL-15 showed a serum half-life profile similar to that of antibody 38B2 in both IV (Figure 36A) and IP (Figure 36B) injections and showed a dose response up to 120 hours after treatment.

Example

[0415] Mechanism of action study in hPDL1 / PD1 transgenic BALB / c mice with hPDL1-CT26 tumors 0.5×10 6 Individual hPDL1 / CT26 cells were subcutaneously inoculated into the lower right abdominal area of hPD1 / PDL1 transgenic BALB / c mice. When the tumor size reached approximately 175 mm 3When reaching, mice were intravenously injected with 0.06 or 0.35 μmol / kg each of (1) N65S-1N-38B2 / IL-1, (2) N65S-1N-DP47 / IL-15 (non-targeted control), (3) antibody 38B2, and (4) the combination of antibody 38B2 and N65S-1N-38B2 / IL-15. Tumor size was measured twice a week, and the mice were sacrificed on the 7th day after dosing (Figure 37A). Blood and draining lymph nodes (DLN) were collected from all groups for flow cytometry. Tumors were collected for TIL analysis (Figure 37B). One-way ANOVA was used for statistical analysis. When a significant F-statistic (the ratio of treatment variance to error variance) was obtained, comparisons between different groups were performed using the Games-Howell (assuming unequal variances) or Tukey (assuming equal variances) test. Comparison between vehicle and other groups, *p<0.05; **p<0.01; ***p<0.001. Error bars represented the standard error of the mean (SEM).

[0416] N65S-1N-38B2 / IL-15 was shown to bind to TILs expressing PD1 and IL2Rβγ and differentially expand CD8+ T cells. Specifically, when analyzing the CD8 / CD4 ratio in tumors, blood, and draining lymph nodes (treated with antibody (1) 38B2, (2) the PD1-targeted fusion protein N65S-1N-38B2 / IL-15, (3) the non-targeted fusion protein N65S-1N-DP47 / IL-15, or (4) the combination of 38B2 and N65S-1N-38B2 / IL-15), it was found that N65S-1N-38B2 / IL-15 promoted CD8 + T cell expansion in tumors but not in blood and draining lymph nodes. The CD8 / CD4 ratio did not change as a result of other treatments ((1) 38B2, (2) the non-targeted fusion protein N65S-1N-DP47 / IL-15, or (3) the combination of 38B2 and N65S-1N-38B2 / IL-15) (Figure 37C).

[0417] Next, CD8 and CD4 subtypes in tumors (treated with tumor (1) antibody 38B2, (2) PD1-targeted fusion protein N65S-1N-38B2 / IL-15, (3) non-targeted fusion protein N65S-1N-DP47 / IL-15, or (4) combination of 38B2 and N65S-1N-38B2 / IL-15) were analyzed. Effector memory CD8 T cells in tumors were found to increase significantly for N65S-1N-38B2 / IL-15-treated samples and slightly for 38B2-treated samples (Figure 37D). No differences were observed among other T cell subtypes in different treatment groups.

Example

[0418] N65S-1N-m3A7 / IL-15 (substitute) efficacy study in multiple mouse syngeneic tumor models The indicated amount of tumor cells (Table 24) in 0.1 mL of PBS was subcutaneously inoculated into the right flank of mice. The animals were randomized, and treatment was initiated when the average tumor volume reached approximately 100 mm 3 (between 75 and 125 mm 3 ). Tumor volume was measured twice a week in two dimensions using calipers, and the volume was expressed in mm3 using the formula: V = (L × W × W) / 2, where V is the tumor volume, L is the length of the tumor (longest tumor dimension), and W is the width of the tumor (longest tumor dimension perpendicular to L).

[0419] Significant antitumor efficacy was observed in all 12 models with single-dose treatment (TGI > 50%) (Table 24). Complete responses (no tumors) were seen for CT26, EMT6, MC38, H22, A20, and Pan02 (Table 24). Data for EMT6 shown as an example (Figure 38).

[0420]

Table 24

[0421]

Table 25-1

Table 25-2

Table 25-3

Table 25-4

Table 25-5

Table 25-6

Table 25-7

Table 25-8

Table 25-9

Table 25-10

Table 25-11

Table 25-12

Table 25-13

Table 25-14

Table 25-15

Table 25-16

Table 25-17

Table 25-18

[0422]

Table 26

[0423]

Table 27

[0424]

Table 28-1

Table 28-2

Table 28-3

Table 28-4

Table 28-5

Table 28-6

Claims

1. An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, each of the heavy and light chain variable regions comprising CDR1, CDR2 and CDR3, wherein: The sequence of CDR1 of the heavy chain variable region (CDR1H) comprises the sequence of SEQ ID NO: 21; The sequence of CDR2 of the heavy chain variable region (CDR2H) comprises the sequence of SEQ ID NO: 22; The sequence of CDR2 of the heavy chain variable region (CDR3H) comprises the sequence of SEQ ID NO: 23; The sequence of CDR1 of the light chain variable region (CDR1L) is １３ X １４ X １５ X １６ IX １７ X １８ WX １９ X ２０ (SEQ ID NO: 135); X １３ is A or V; X １４ is S or G; X １５ is Q, E or R; X １６ is G, S, D or N; X １７ is G, S or N; X １８ is S, I, R, T, K, P, N, H or V; X １９ is L or V; X ２０ is G or A; The sequence of CDR2 of the light chain variable region (CDR2L) is sequence X ２１ AX ２２ X ２３ X ２４ X ２５ X ２６ (SEQ ID NO: 136); X ２１ is S, D, E or A; X ２２ is S or K; X ２３ is S, N, T, R or D; X ２４ is L or V; X ２５ is Q, E or H; X ２６ is S, N, A, R, P or T; The sequence of the CDR3 of the light chain variable region (CDR3L) is the sequence QQX ２７ X ２８ SFPX ２９ X ３０ (SEQ ID NO: 137); X ２７ is A or G; X ２８ is N, D or Y; X ２９ is F or L; and X ３０ is A or T, an anti-PD-1 antibody or antigen-binding fragment thereof.

2. The sequence of CDR1H comprises the sequence of SEQ ID NO:21; The sequence of CDR2H comprises the sequence of SEQ ID NO: 22; The sequence of CDR3H comprises the sequence of SEQ ID NO: 23; The sequence of CDR1L comprises the sequence of SEQ ID NO:96; The sequence of CDR2L comprises the sequence of SEQ ID NO:97; The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, wherein the sequence of CDR3L comprises the sequence of SEQ ID NO:

82.

3. a. the heavy chain variable region comprises SEQ ID NO:24 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24; b. the light chain variable region comprises SEQ ID NO:98 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:98; The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 2.

4. a. The sequences of CDR1H, CDR2H and CDR3H are SEQ ID NOs: 21, 22 and 2 Including 3; b. The sequences of CDR1L, CDR2L and CDR3L are i. SEQ ID NOs: 45, 46 and 47; ii. SEQ ID NOs: 66, 67 and 68; iii. SEQ ID NOs: 70, 67 and 71; iv. SEQ ID NOs: 73, 74 and 75; v. SEQ ID NOs: 77, 78 and 47; vi. SEQ ID NOs: 80, 81 and 82; vii. SEQ ID NOs: 77, 78 and 84; viii. SEQ ID NOs: 77, 86 and 47; ix. SEQ ID NOs: 88, 89 and 47; x. SEQ ID NOs: 66, 67 and 47; xi. SEQ ID NOs: 80, 92 and 75; xii. SEQ ID NOs: 80, 94 and 71; xiii. SEQ ID NOs: 99, 100 and 47; xiv. SEQ ID NOs: 102, 103 and 104; xv. SEQ ID NOs: 106, 103 and 47; xvi. SEQ ID NOs: 108, 103 and 47; xvii. SEQ ID NOs: 110, 111 and 75; xviii. SEQ ID NOs: 77, 103 and 113; xix. SEQ ID NOs: 77, 111 and 47; xx. SEQ ID NOs: 116, 67 and 47; xxi. SEQ ID NOs: 118, 119 and 47; xxii. SEQ ID NOs: 80, 78 and 47; xxiii. SEQ ID NOs: 122, 103 and 47; xxiv. SEQ ID NOs: 124, 125 and 75; xxv. SEQ ID NOs: 127, 38 and 68; xxvi. SEQ ID NOs: 129, 130 and 47; or SEQ ID NOs: 132, 133 and 75 The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1,

5. a. The heavy chain variable region comprises: i. SEQ ID NO: 24, or ii. a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24 Including; b. The light chain variable region comprises: i. SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131 or 134, or ii. a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 48, 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131, or 134. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 4.

6. An anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, each of the heavy and light chain variable regions comprising CDR1, CDR2 and CDR3, wherein: The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L is the sequence RSSX １ SLLX ２ SNGX ３ X ４ YLD (SEQ ID NO: 62), X １ is Q or E; X ２ is H or Y; X ３ is Y or N; X ４ is T or N; The sequence of CDR2L is sequence X ５ X ６ S.X. ７ X ８ X ９ X １０ (SEQ ID NO: 63), X ５ is L, Q or E; X ６ is S, A or V; X ７ is H, N, T or S; X ８ is R or L; X ９ is G, A or H; X １０ is S or T; The sequence of CDR3L is the sequence MQGX １１ X １２ WPYT (SEQ ID NO:64), X １１ is A, T or S; X １２ is H or R; An anti-PD-1 antibody or an antigen-binding fragment thereof.

7. The sequence of CDR1H comprises the sequence of SEQ ID NO: 17; The sequence of CDR2H comprises the sequence of SEQ ID NO: 18; The sequence of CDR3H comprises the sequence of SEQ ID NO: 19; The sequence of CDR1L comprises the sequence of SEQ ID NO:41; The sequence of CDR2L comprises the sequence of SEQ ID NO:49; The anti-PD-1 antibody or antigen-binding fragment thereof of claim 6, wherein the sequence of CDR3L comprises the sequence of SEQ ID NO:

50.

8. a. the heavy chain variable region comprises SEQ ID NO:20 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20; b. the light chain variable region comprises SEQ ID NO:51 or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:51; The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 7.

9. a. the sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOs: 17, 18 and 19; b. The sequences of CDR1L, CDR2L and CDR3L are i. SEQ ID NOs: 41, 42 and 43; ii. SEQ ID NOs: 41, 52 and 53; iii. SEQ ID NOs: 41, 55 and 56; or iv. SEQ ID NOs: 58, 59 and 60 The anti-PD-1 antibody or antigen-binding fragment thereof of claim 6,

10. a. The heavy chain variable region comprises: i. SEQ ID NO: 20, or ii. Contains a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:

20. fruit; b. The light chain variable region comprises: i. SEQ ID NO: 44, 54, 57 or 61, or ii. A sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 44, 54, 57 or 61. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 9,

11. An anti-PD-1 antibody or antigen-binding fragment thereof that binds to the same epitope as the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which is a chimeric antibody, a CDR-grafted antibody, or a humanized antibody, or an antigen-binding fragment thereof.

13. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is a multispecific or bispecific antibody or antigen-binding fragment thereof.

14. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 13, which is a bispecific antibody comprising a complementary region that binds to PD-L1 or PD-L2.

15. scFv, Fv, Fab', Fab, F(ab') ２ or a diabody.

16. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody is an IgG class immunoglobulin.

17. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 16, having an isotype of IgG4.

18. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, comprising an S241P substitution in the constant region of the heavy chain.

19. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 18, which is deglycosylated.

20. 20. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the heavy chain lacks a C-terminal lysine.

21. 21. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 20, comprising a first and a second heavy chain constant region and comprising at least one modification in the CH3 domain of the first and second heavy chain that causes heterodimerization.

22. 22. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 21, wherein the modification in the CH3 domain of the first heavy chain is distinct from the modification in the CH3 domain of the second heavy chain.

23. 23. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 21 or 22, wherein the first heavy chain comprises an amino acid substitution selected from the group consisting of T350V, L351Y, S354C, S364H, T366Y, T366W, F405A, Y407V (Kabat EU index numbering).

24. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 21 to 23, wherein the hole modification comprises an amino acid substitution selected from the group consisting of Y349C, T350V, T366L, T366S, L368A, K392L, T394W, Y407V, Y407T (Kabat EU index numbering).

25. a. the CH3 domain of the first heavy chain comprises one or more of the amino acid substitutions S354C and T366W and the CH3 domain of the second heavy chain comprises one or more of the amino acid substitutions Y349C, T366S, L368A and Y407V; b. the CH3 domain of the first heavy chain comprises one or more of the amino acid substitutions T350V, L351Y, F405A and Y407V and the CH3 domain of the second heavy chain comprises one or more of the amino acid substitutions T350V, T366L, K392L and T394W; or c. the CH3 domain of the first heavy chain comprises one or more of the amino acid substitutions L351Y, F405A and Y407V, and the CH3 domain of the second heavy chain comprises one or more of the amino acid substitutions T366L, K392L and T394W. (All Kabat EU index numbering), The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 22 to 24.

26. 26. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 25, wherein the Fc domain contains one or more amino acid substitutions that reduce binding to an Fc receptor or reduce effector function.

27. 27. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 26, wherein the one or more amino acid substitutions are at one or more positions selected from the group of L234, L235, and P329 (Kabat EU index numbering).

28. 28. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 27, wherein the one or more amino acid substitutions are selected from L234A, L235A, and P329G (Kabat EU index numbering).

29. A fusion protein comprising: a. an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, each of the heavy chain and light chain variable regions comprising CDR1, CDR2 and CDR3; b. an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO:214, or an amino acid sequence that is at least 95% identical to SEQ ID NO:214; and c. an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO:212 or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO:212; d. A first linker polypeptide that connects the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide. A fusion protein comprising:

30. The fusion protein of claim 29, wherein the IL-15Rα sushi domain polypeptide is fused to the N-terminus of the IL-15 polypeptide.

31. The fusion protein of claim 29 or 30, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a constant region, and the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide are fused to the C-terminus of the constant region.

32. The fusion protein of claim 29 or 30, wherein the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide are fused to the N-terminus of the heavy chain variable region.

33. The fusion protein of claim 32, wherein the second linker joins (i) the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide and (ii) the N-terminus of the heavy chain variable region.

34. The fusion protein of any one of claims 29 to 33, wherein the first linker is between 25 and 35 amino acids in length.

35. 35. The fusion protein of claim 34, wherein the first linker consists essentially of Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L) and Gln (Q).

36. The fusion protein of any one of claims 29 to 33, wherein the first linker comprises a sequence selected from the group consisting of SEQ ID NOs: 215-218 and 244.

37. The fusion protein of claim 36, wherein the first linker comprises the sequence of SEQ ID NO:

215.

38. The fusion protein of any one of claims 33 to 37, wherein the second linker is between 25 and 35 amino acids in length.

39. 39. The fusion protein of claim 38, wherein the first linker consists essentially of Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L) and Gln (Q).

40. The fusion protein of any one of claims 33 to 37, wherein the second linker comprises a sequence selected from the group consisting of SEQ ID NOs: 215-218 and 244.

41. The fusion protein of claim 40, wherein the second linker comprises the sequence of SEQ ID NO:

216.

42. The fusion protein of any one of claims 29 to 41, wherein the fusion protein comprises no more than one IL-15Rα sushi domain polypeptide and no more than one IL-15 polypeptide.

43. The fusion protein according to any one of claims 29 to 42, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 28.

44. The fusion protein of any one of claims 29 to 43, wherein the IL-15 polypeptide is a mutant IL-15 polypeptide comprising one or more amino acid substitutions at positions 45, 65 or 108 as numbered according to the mature human IL-15 sequence of SEQ ID NO:

212.

45. The fusion protein of claim 44, wherein the one or more amino acid substitutions in the IL-15 polypeptide are selected from L45A, N65A, N65D, N65S, N65K and Q108S as numbered according to the mature human IL-15 sequence of SEQ ID NO:

212.

46. The IL-15Rα sushi domain polypeptide is the mature human IL-15Rα sushi domain polypeptide of SEQ ID NO:

213.

46. ​​The fusion protein of any one of claims 29 to 45, comprising an amino acid substitution at position 60 as numbered according to the -15Rα sushi domain sequence.

47. The fusion protein of claim 46, wherein the amino acid substitution at position 60 is an N60A substitution.

48. A light chain sequence comprising sequence number 219 or SEQ ID NO:98, and: a. a first heavy chain sequence comprising SEQ ID NO:220 and a second heavy chain sequence comprising SEQ ID NO:224; or a first heavy chain sequence comprising SEQ ID NO:221 and a second heavy chain sequence comprising SEQ ID NO:225; or b. a first heavy chain sequence comprising SEQ ID NO:222 and a second heavy chain sequence comprising SEQ ID NO:226; or a first heavy chain sequence comprising SEQ ID NO:223 and a second heavy chain sequence comprising SEQ ID NO:

227.

30. The fusion protein of claim 29, comprising:

49. A light chain sequence comprising SEQ ID NO:219 or SEQ ID NO:98, and: a. a first heavy chain sequence comprising SEQ ID NO:220 and a second heavy chain sequence comprising SEQ ID NO:228; or a first heavy chain sequence comprising SEQ ID NO:221 and a second heavy chain sequence comprising SEQ ID NO:229; b. a first heavy chain sequence comprising SEQ ID NO:222 and a second heavy chain sequence comprising SEQ ID NO:230; or a first heavy chain sequence comprising SEQ ID NO:223 and a second heavy chain sequence comprising SEQ ID NO:231; c. a first heavy chain sequence comprising SEQ ID NO:220 and a second heavy chain sequence comprising SEQ ID NO:236; or a first heavy chain sequence comprising SEQ ID NO:221 and a second heavy chain sequence comprising SEQ ID NO:237; d. a first heavy chain sequence comprising SEQ ID NO:222 and a second heavy chain sequence comprising SEQ ID NO:238; or a first heavy chain sequence comprising SEQ ID NO:223 and a second heavy chain sequence comprising SEQ ID NO:239; e. a first heavy chain sequence comprising SEQ ID NO:240 and a second heavy chain sequence comprising SEQ ID NO:241; or f. The fusion protein of claim 29, comprising a first heavy chain sequence comprising SEQ ID NO:240 and a second heavy chain sequence comprising SEQ ID NO:

242.

50. A light chain sequence comprising SEQ ID NO:219 or SEQ ID NO:98, and: a. a first heavy chain sequence comprising SEQ ID NO:220 and a second heavy chain sequence comprising SEQ ID NO:232; or a first heavy chain sequence comprising SEQ ID NO:221 and a second heavy chain sequence comprising SEQ ID NO:233; b. a first heavy chain sequence comprising SEQ ID NO:222 and a second heavy chain sequence comprising SEQ ID NO:234; or a first heavy chain sequence comprising SEQ ID NO:223 and a second heavy chain sequence comprising SEQ ID NO:235; or c) The fusion protein of claim 29, comprising a first heavy chain sequence comprising SEQ ID NO:240 and a second heavy chain sequence comprising SEQ ID NO:

243.

51. A nucleic acid sequence encoding the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 28, or a fusion protein of any one of claims 29 to 50.

52. A vector comprising the nucleic acid of claim 51.

53. 53. A cell comprising the nucleic acid of claim 51 or the vector of claim 52.

54. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, A cell expressing the fusion protein according to any one of claims 29 to 47.

55. A T cell expressing a PD-1 binding protein comprising the CDR of the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

56. A T cell expressing a PD-1 binding protein comprising a heavy and light variable chain of any one of claims 1 to 10.

57. 51. The anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 28, or the fusion protein of any one of claims 29 to 50, wherein the antibody or antigen-binding fragment or fusion protein is conjugated to one or more of a cytotoxin, a fluorescent label, and an imaging agent.

58. A pharmaceutical composition comprising: (i) the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1 to 28; or the fusion protein of any one of claims 29 to 50; and (ii) a pharma- ceutical acceptable carrier.

59. 51. A method of inhibiting binding of PD-1 to a ligand of PD-1 in a subject in need thereof, comprising administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-28, or a fusion protein of any one of claims 29-50.

60. The method of claim 59, wherein the ligand of PD-1 is PD-L1 or PD-L2.

61. 51. A method of increasing T cell activation in a subject in need thereof, comprising administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-28, or a fusion protein of any one of claims 29-50.

62. 51. A method of stimulating the immune system in a subject in need thereof, comprising administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-28, or a fusion protein of any one of claims 29-50.

63. 63. The method of claim 62, wherein the subject has upregulated expression of PD-L1 or the subject has been identified as positive for expression of PD-L1.

64. 51. A method of reducing viral replication in a subject in need thereof, comprising administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-28, or a fusion protein of any one of claims 29-50.

65. 51. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-28, or a fusion protein of any one of claims 29-50.

66. 66. The method of claim 65, wherein the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cell carcinoma, prostate cancer, breast cancer, bladder cancer or gastric cancer.

67. A method for reducing tumor growth in a subject in need thereof, comprising administering to the subject a compound according to any one of claims 1 to 4.

28. A method comprising administering an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 29 to 50, or a fusion protein of any one of claims 29 to 50.

68. 51. A method of reducing tumor metastasis in a subject in need thereof, comprising administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-28, or a fusion protein of any one of claims 29-50.

69. 69. The method of any one of claims 59-68, which is unresponsive to therapy with a checkpoint inhibitor, or which initially responds to checkpoint inhibitor treatment but later becomes resistant to checkpoint inhibitor blockade.

70. 69. The method of any one of claims 59 to 68, further comprising administering an additional therapeutic agent or therapy.

71. 71. The method of claim 70, wherein the additional therapeutic agent is selected from the group consisting of a cancer vaccine, a checkpoint inhibitor, an antibody against a tumor-specific antigen, a Bacillus Calmette-Guerin (BCG) vaccine, a cytotoxin, an interleukin-6 receptor (IL-6R) inhibitor, an interleukin-4 receptor (IL-4R) inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, an antibody-drug conjugate, an anti-inflammatory drug, and a dietary supplement.

72. 72. The method of claim 71, wherein the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1 or PD-L2 inhibitor.

73. 71. The method of claim 70, wherein the additional therapeutic agent is an inhibitor of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226 or CD160.

74. 71. The method of claim 70, wherein the therapy is chemotherapy, radiation therapy or surgery.

75. The method of any one of claims 70-74, wherein the additional therapeutic agent or therapy is administered simultaneously or sequentially with the anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein.

76. The method of any one of claims 70-73, wherein the additional therapeutic agent is administered separately or as a mixture with the anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein.

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