Anti-PD-1 antibodies and fusion proteins

Abstract

Provided herein are recombinant antibodies, antigen-binding fragments and fusion proteins thereof, nucleic acid molecules encoding same and therapeutic compositions thereof useful for binding to and inhibiting programmed death 1 (PD-1), as well as methods of using such antibodies, including methods of enhancing T cell and NK cell function to increase cell- and cytokine-mediated immunity and methods of treating various immune dysfunction-associated disorders, including cancer and infectious diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 043,114, filed June 23, 2020, and to U.S. Provisional Patent Application No. 63 / 111,459, filed November 9, 2020, the entireties of which are incorporated herein by reference.

[0002] The present disclosure relates generally to the fields of molecular biology and medicine. More specifically, the present disclosure provides fusion proteins comprising an anti-PD-1 antibody or antigen-binding fragment thereof linked to an IL-15 polypeptide, which is in turn linked to an IL-15 receptor alpha (IL-15Rα) polypeptide comprising an IL-15Rα sushi domain. Furthermore, the present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to PD-1, as well as fusion proteins comprising such anti-PD-1 antibodies and PD-1-binding fragments thereof. Therapeutic compositions comprising the antibody fusions or antibodies for treating diseases are also disclosed. [Background technology]

[0003] PD-1 is a 55-kD type I transmembrane protein of 286 amino acids containing 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, with the nearest tyrosine (VAYEEL in mouse PD-1) located within the ITIM. Human and mouse PD-1 proteins share approximately 60% amino acid identity, and four potential N-glycosylation sites and residues defining the Ig-V domain are conserved. The ITIM-like motif surrounding the ITIM in the cytoplasmic region and the C-terminal tyrosine are also conserved between human and mouse orthologs. PD-1 is a member of the CD-28 family of receptors and is primarily expressed on mature T cells in peripheral tissues and the tumor microenvironment. PD-1 is also expressed on B cells, professional antigen-presenting cells (APCs), and other non-T cell subsets, including natural killer (NK) cells.

[0004] PD-1 ligands 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 primarily expressed on both lymphoid and non-lymphoid tissues, such as CD4 and CD8 T cells, macrophage-lineage cells, and peripheral tissues, as well as on tumor cells, virus-infected cells, and autoimmune tissue cells. However, PD-L2 has more restricted expression than PD-L1, being expressed only on macrophages and activated dendritic cells. PD-1 ligands are expressed in numerous 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 inducible in almost all cancers. Interaction between PD-1 and its ligand results in the dephosphorylation and inactivation of the T cell kinase ZAP70 and the recruitment of SHP2. SHP2 directly dephosphorylates PI3K and inhibits downstream activation of Aktin, resulting in a reduction in tumor-infiltrating lymphocytes and reduced T cell receptor-mediated proliferation, leading to immune evasion. Blocking the interaction between PD-1 and PD-L1 reverses immune suppression, and the effect can be additive when the interaction of PD-1 with PD-L2 is similarly blocked.

[0005] Several commercially available anti-PD-1 antibodies are currently used to treat diseases involving PD-1-mediated immunosuppression. However, only a subset of patients respond adequately to these therapies. Depending on the indication and other factors, the typical response rate for 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), which contains activated anti-tumor CD8+ and CD4+ T cells and other effectors, such as NK cells.

[0006] Combination therapies involving PD-1 antibodies 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 systemic toxicity associated with both the high circulating concentrations of cytokines required for efficacy and the lack of specificity of the administered cytokines for affected cells and tissues.

[0007] IL-15 is a 12.5-kD glycoprotein with 114 amino acids and belongs to the cytokine 4 α-helical bundle family, which 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 T cells to exert immune responses without modulating the effects of 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 soluble or membrane-bound. The major form of IL-15, membrane-bound IL-15, is formed by direct binding of IL-15 to the cell membrane or by 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 normally forms a complex with the IL-15 receptor α expressed on APCs before binding to the functional IL-15Rβ and γ units on T cells and NK cells. IL-15 binds to the IL-15Rα receptor alone with high affinity (Ka=1.1011 M -1 ) can bind to the IL-15Rβγc signaling complex with lower affinity (Ka=1.109M -1The sushi domain (29.5 kD) of IL-15Rα plays an important role in the complex formation between IL-15 and IL-15Rα.

[0009] One of the limitations of systemic IL-15 treatment is its extremely short in vivo half-life. Therefore, there is a need to generate suitable immunostimulatory forms of IL-15 / IL-15Rα with a longer in vivo half-life while retaining its ability to modulate immune responses. Furthermore, there is a need for effective IL-15 antagonists that can be selectively targeted to disease sites to avoid unwanted systemic toxicity and provide more effective therapeutic benefit. Summary of the Invention [Problem to be solved by the invention]

[0010] Compounds that modulate PD-1 activity have potential as therapeutic agents for the treatment of various diseases and disorders, including cancer, inflammation, and autoimmune diseases. There is a significant unmet need to develop novel strategies for targeting various effector molecules to disease sites to provide therapeutic benefit without the side effects associated with nonspecific immune activity. [Means for solving the problem]

[0011] Provided herein are fusion proteins, antibodies, and antigen-binding fragments thereof that bind to PD-1. In embodiments, the fusion proteins comprise an anti-PD-1 antibody or 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 disclosure provides a fusion protein comprising a PD-1 binding protein, an IL-15 receptor (IL-15R) binding protein (e.g., IL-15 or an IL-15R-binding portion thereof), and an IL-15 binding protein (e.g., IL-15Rα or an IL-15-binding portion thereof comprising the IL-15Rα sushi domain). In one aspect, the PD-1 binding protein is an antibody or PD-1-binding fragment thereof. In one embodiment, the IL-15 receptor-binding portion is IL-15. In embodiments, IL-15 is linked directly or indirectly 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, wherein the C-terminus of the IL-15 polypeptide is covalently linked, either directly or via a polypeptide linker, to the N-terminus of the heavy chain variable region of one of the antibody heavy chains (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 an IL-15Rα sushi domain polypeptide.

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

[0015] In embodiments in which 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 of the heavy chain fusion with the heavy chain lacking the fusion). In embodiments, an amino acid residue in the CH3 domain (CH3-1) of one heavy chain contains an amino acid substitution that replaces the amino acid with an amino acid residue having a larger side chain volume, thereby generating a "knob" in the CH3 domain that can be positioned into a "hole" created by replacing an amino acid residue with an amino acid residue having a smaller side chain volume present in the CH3 domain (CH3-2) of the other heavy chain. In embodiments, the amino acid substitution is selected from the following group: (1) First heavy chain CH3 domain: S354C, T366W; second heavy chain CH3 domain: 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 second heavy chain is linked to an IL-15 / IL-15Rα sushi domain.

[0017] In embodiments, the IL-15Rα polypeptide and the IL-15 polypeptide are connected by a first linker. In embodiments, the IL-15 polypeptide and the anti-PD-1 antibody, or antigen-binding portion thereof, are connected by a second linker. In embodiments, the linker length can independently be between 10 and 40 amino acids. In embodiments, the linker length is between 25 and 35 amino acids. In embodiments, the linker sequence comprises adjacent neutral amino acids selected from the group including Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L), and Gln (Q), most preferably from the group including 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 comprises 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 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 L45A, N65S / A / D / K, and / or Q108S substitutions in IL-15. In one embodiment, the amino acid substitution is an N65S substitution in IL-15.

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

[0020] In embodiments, the fusion protein comprises an IL-15 polypeptide comprising one of the following amino acid substitutions: N65S, N65A, or N65D. In embodiments, 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 provides a method for manufacturing a semiconductor device comprising: (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; (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 connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide or a derivative thereof; The present invention relates to a fusion protein comprising:

[0022] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; (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 at least 95% identical to SEQ ID NO: 214; and (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; The present invention relates to a fusion protein comprising:

[0023] The anti-PD-1 antibody or antigen-binding fragment thereof, portion of the fusion protein, can be any antibody or antigen-binding fragment thereof that specifically binds to PD-1, including those comprising the corresponding heavy and light chain variable regions and / or CDRs provided in Figure 1 or otherwise described herein. In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the CDRs of the heavy and light chain variable regions comprise the following sequences: the sequence of CDR1 (CDR1H) of the heavy chain variable region comprises the sequence of SEQ ID NO: 21; the sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence of SEQ ID NO: 22; the sequence of CDR2 (CDR3H) of the heavy chain variable region comprises the sequence of SEQ ID NO: 23; The sequence of CDR1 (CDR1L) of the light chain variable region is 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 (CDR2L) of the light chain variable region is 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 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:24 or a sequence 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 SEQ ID NO:98 or a sequence 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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 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 NO: 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.

[0027] In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:24 or a sequence 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 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 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 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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), X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L is X5X6SX7X8X9X 10 (SEQ ID NO: 63), 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 MQGX 11 X 12 WPYT (SEQ ID NO: 64), X 11is A, T or S; X 12 is H or R.

[0029] In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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 the CDR3 (CDR3L) of the light chain variable region comprises the sequence of SEQ ID NO:50.

[0030] In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:20 or a sequence 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 SEQ ID NO:51 or a sequence 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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: 17, 18 and 19, respectively; The sequences of CDR1L, CDR2L and CDR3L 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, respectively.

[0032] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:20 or a sequence 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 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 NOs: 44, 54, 57 or 61.

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

[0034] 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 is 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 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 CDR3L is 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 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 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.

[0036] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the following: a heavy chain variable region comprising SEQ ID NO:24 or a sequence 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 Includes:

[0037] In embodiments, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof 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 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 NO: 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.

[0038] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the following: a heavy chain variable region comprising SEQ ID NO:24 or a sequence 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: 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 NOs: 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. Includes:

[0039] 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: 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), X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L is X5X6SX7X8X9X 10 (SEQ ID NO: 63), 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 MQGX 11 X 12 WPYT (SEQ ID NO: 64), X 11 is A, T or S; X 12 is H or R.

[0040] 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: 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.

[0041] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the following: a heavy chain variable region comprising SEQ ID NO:20 or a sequence 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. Includes:

[0042] In embodiments, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof comprise the following sequences: the sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOs: 17, 18 and 19, respectively; The sequences of CDR1L, CDR2L and CDR3L 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, respectively.

[0043] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the following: a heavy chain variable region comprising SEQ ID NO:20 or a sequence 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 NOs: 44, 54, 57 or 61 Includes:

[0044] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

[0045] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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).

[0046] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions T366L, K392L and T394W (Kabat EU index numbering).

[0047] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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, optionally wherein 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 amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

[0048] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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, optionally wherein 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 amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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).

[0049] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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: 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, optionally wherein 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 amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions T366L, K392L and T394W (Kabat EU index numbering).

[0050] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 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 at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

[0051] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 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 at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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).

[0052] In one aspect, the present disclosure provides a method for manufacturing a method of a cellular membrane comprising: (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; 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; 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 at least 95% identical to SEQ ID NO: 214; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions T366L, K392L and T394W (Kabat EU index numbering).

[0053] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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; 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 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 amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

[0054] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 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 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 amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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).

[0055] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (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; 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 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 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 amino acid N28 of SEQ ID NO: 214 is substituted with A; (iv) a first linker polypeptide connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; In 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 contains the amino acid substitutions T366L, K392L and T394W (Kabat EU index numbering).

[0056] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof provided by the present disclosure is a multispecific or bispecific antibody or antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment is a bispecific antibody that contains complementary regions that bind to PD-L1 or PD-L2. The present disclosure also provides conjugates or fusion molecules of the antibody to, for example, but not limited to, a fluorescent label, an imaging agent, a therapeutic agent, or a cytotoxic agent.

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

[0058] In embodiments, the present disclosure provides nucleic acid molecules encoding the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein and nucleic acid molecules encoding the fusion proteins disclosed herein, as well as vectors comprising such nucleic acid molecules. Also provided are cells comprising vectors encoding the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein or the fusion proteins disclosed herein.

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

[0060] In embodiments, the present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-PD-1 antibody or antigen-binding fragment thereof, or fusion protein disclosed herein, 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 antigen-binding fragment thereof, or fusion protein 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 antigen-binding fragment thereof, or fusion protein 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 antigen-binding fragment thereof, or fusion protein 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, comprising administering to a subject in need thereof an effective amount of an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein. The present disclosure further provides a method of inhibiting PD-1-mediated immunosuppression 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, or fusion protein disclosed herein.

[0063] The present disclosure further provides a method of stimulating an immune response against cells or tissues that express 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, or fusion protein disclosed herein. In certain embodiments, the PD-1-expressing cells or tissues are cancerous cells or cells infected with a pathogen, including, but not limited to, a virus or bacteria.

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

[0065] In another aspect, the disclosure provides a method of increasing 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 antigen-binding fragment thereof, or fusion protein disclosed herein, or a pharmaceutical composition disclosed herein.

[0066] In another aspect, the disclosure provides methods of treating a condition in a subject that would benefit from upregulation of the subject's immune response, comprising administering to the subject an effective amount of an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein 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 methods of using an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein to treat a subject who does not respond to therapy with a checkpoint inhibitor (primary resistance) and / or to treat a subject who initially responds to checkpoint inhibitor treatment but later becomes resistant to checkpoint inhibitor blockade (secondary or acquired resistance). Such methods of treatment comprise administering to the subject an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein 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 antigen-binding fragment thereof, or fusion protein disclosed herein to a subject in need thereof, further comprising administering to the subject an additional therapeutic agent or therapy, where the additional therapeutic agent or therapy is selected from the group consisting of a cancer vaccine, a checkpoint inhibitor, an antibody against a tumor-specific antigen, Bacillus Calmette-Guérin (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. In an embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein to a subject in need thereof, further comprising administering to the subject a checkpoint inhibitor selected from a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor. In embodiments, the present disclosure provides methods of administering an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein to a subject in need thereof, further comprising administering to the subject one or more of an inhibitor of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, and / or CD 160. In embodiments, the present disclosure provides methods of administering an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein to a subject in need thereof, further comprising administering an additional therapeutic agent or therapy, 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 disclosed herein. In embodiments, the present disclosure provides methods of administering an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein to a subject in need thereof, further comprising administering to the subject an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is administered separately or in admixture with the anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein.In one embodiment, the present disclosure provides a method of administering an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein disclosed herein in combination with chemotherapy, radiation therapy, or surgery. [Brief explanation of the drawings]

[0069] [Figure 1-1]Figure 1 shows the heavy and light chain variable domain sequences and CDR sequences of the anti-PD-1 antibodies disclosed herein. CDR sequences are according to Kabat (CDR sequences according to AbM / Chothia exclude underlined amino acids). Figure 1A provides the heavy and light chain variable regions of six anti-human PD-1 (hPD-1) antibodies identified by library screening. Figure 1B provides the light chain and CDR sequences of 23H9 and of 23H9 variants identified by light chain shuffling, in which a library of light chains was paired with the 23H9 heavy chain sequence. X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; 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; X10 is S or T; X11 is A, T or S; and X12 is H or R. Figure 1C provides the light chain and CDR sequences of 24H9 and of 24H9 variants identified by light chain shuffling, where a library of light chains was paired with the 24H9 heavy chain sequence. X13 is A or V; X14 is S or G; X15 is Q, E or R; X16 is G, S, D or N; X17 is G, S or N; X18 is S, I, R, T, K, P, N, H or V; X19 is L or V; X20 is G or A; X21 is S, D, E or A; X22 is S or K; X23 is S, N, T, R or D; X24 is L or V; X25 is Q, E or H; X26 is S, N, A, R, P or T; X27 is A or G; X28 is N, D or Y; X29 is F or L; and X30 is A or T. Figure ID provides the sequences of hPD-1 / mPD-1 that cross-react with antibody R3A9 as well as R3A9 derivatives identified by light chain shuffling, in which a library of light chains was paired with the R3A9 heavy chain sequence. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 2]Figure 2 illustrates the binding of antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 to hPD-1-Fc (Figure 2A, labeled as in Figure 2B) and to hPD-1-his (Figure 2B) as determined by ELISA. [Figure 3] Figure 3 shows the ability of antibodies 19B6, 19B10, 23A8, 23H9, 24H9 and 23A11 to block the interaction between hPD-1 and hPD-L1. [Figure 4] Figures 4A-C illustrate the binding of antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 to hPD-1-293 cells (Figure 4A), SEB-activated PBMCs from donor "EA" (low PD-1 expression) (Figure 4B), and SEB-activated PBMCs from donor "AF" (high PD-1 expression) (Figure 4C). Figure 4A: EC50 23A8>23H9>19B10. MFI 23A8>23H9>19B10. Figure 4B: 23H9>24H9 and 23A8. Figure 4C: 23H9>24H9 and 23A8. [Figure 5] Figure 5 illustrates the ability of antibodies 23A8 and 23H9 (Figure 5A) and 19B6 and 24H9 (Figure 5B) to block the interaction between PD-1 and hPD-L1. [Figure 6-1] Figure 6 shows the binding of antibodies 23H9, 31B1, 33C4, 33G8, and 34C1 to hPD-1-Fc (Figure 6A, identical representation to Figure 6B), to biotin-hPD-1-his captured by streptavidin (Figure 6B), to hPD-1-his captured by αhis (Figure 6C, identical representation to Figure 6D), and to blocking of the hPD-1-Fc / hPD-L1-Fc interaction (Figure 6D). No significant differences were observed between the parent antibody 23H9 and its derivatives (Figures 6A and 6B). All derivatives had lower EC50s and higher inhibition values ​​than the parent antibody (Figures 6C and 6D). [Figure 6-2] Continued from Figure 6-1. [Figure 7]Figure 10 shows the binding of antibodies 24H9, 32A11, 32D2, 32D11, 38A6, 38A10, 38A11, 38B2, 38C11, 39B3, 39B11, 39G8, and 38H11 to hPD-1-Fc (first, left-most column), to hPD-1-his captured by streptavidin (second column), to hPD-1-his captured by αhis (third column), and blocking of the hPD-1 / hPD-L1 interaction (fourth column). [Figure 8-1] Figure 8A shows binding of antibodies 31B1, 33C4, 23H9, 38A6, 24H9, and 38B2 to hPD-1-293 cells (Figure 8A, same representation as Figure 8B) and hPD-1 Jurkat cells (Figure 8B), as well as to SEB-activated PBMCs from donor FA (Figure 8C, same representation as Figure 8D) and donor FB (Figure 8D). Figure 8A. MFI of hPD1-HEK293: 24H9 derivative (38B2 and 38A6 > 24H9) > 23H9 derivative (31B1 and 33C4 > 23H9). Figure 8B. MFI of hP01-Jurkat: 23H9 derivative (33C4 > 31B1 > 23H9) > 24H9 derivative (38B2 > 38A6 > 24H9). Figures 8C and 8D. EC50 values ​​for binding to SEB-activated human PBMCs from two donors: 23H9 derivatives (31B1 identical to 33C4) < 24H9 derivatives (38A6 identical to 31B1). MFI for binding to SEB-activated human PBMCs: 23H9 derivatives (31B1 identical to 33C4) > 24H9 derivatives (38A6 identical to 31B1). [Figure 8-2] Continued from Figure 8-1. [Figure 9]Figure 9 illustrates the ability of antibodies 23H9, 31B1, 33C4, 24H9, 38B2, and 38A6 to block the PD-1 / PD-L1 interaction using a Promega binding assay. Figure 9A. EC50 for 23H9 and its derivatives 31B1 and 33C4: 31B1 (0.139 μg / ml) > 33C4 (0.165 μg / ml) > 23H9 (0.511 μg / ml). Maximum fold induction (luminescence): 31B1 (6.48) > 33C4 (6.24) > 23H9 (5.56). Figure 9B. EC50 for 24H9 and its derivatives 38A6 and 38B2: 38B2 (0.855 μg / ml) > 38A6 (0.164 μg / ml) > 24H9 (0.855 μg / ml). Maximum induction factor (luminescence): 38B2 (6.54) > 38A6 (5.66) > 24H9 (4.97). [Figure 10-1] Figures 10A and 10C show IL-2 accumulation in PBMCs from donor FA (Figure 10A) and donor EA (Figure 10B) in the presence of antibodies 23H9, 31B1, 24H9, and 38B2. Figure 10A shows IL-2 accumulation in PBMCs from donor FA: IL-2 concentrations increased dose-dependently in the presence of the indicated antibodies. Figure 10B shows 23H9 and 31B1: EC50 values ​​were 0.344 and 0.107 nM, respectively; IL-2 concentrations at 10 nM antibody concentration were 3.9 and 5.81 μg / ml, respectively. Figure 10C shows 24H9 and 38B2: EC50 values ​​were >10 nM and 0.019 nM, respectively; IL-2 concentrations at 10 nM antibody concentration were 4.5 and 5.0 μg / ml, respectively. [Figure 10-2] Continued from Figure 10-1. [Figure 11] FIG. 1 illustrates the thermal stability measurements of antibodies 780_38 (31B1), 780_39 (33C4), 780_46 (38A6) and 780_54 (38B2) as determined by differential scanning calorimetry (DSC). [Figure 12-1]Figure 12A shows the binding of antibodies 31B1, 33C4, 38B2, and 38A6 to hPD-1FC (Figure 12A), to biotin-labeled hPD-1his captured by streptavidin (Figure 12B, same label as Figure 12A), and to hPD-1his captured by anti-his antibody (Figure 12C, same label as Figure 12A), and blocking of hPD-1-hPD-L1 (Figure 12D) and hPD-1-hPD-L2 interactions (Figure 12E). [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 13-1] Figures 13A-13D show dose-response binding of antibodies 31B1, 33C4, 38A6, and 38B2 to hPD-1-293 cells (Figure 13A), hPD-1-Jurkat cells (Figure 13B), SEB-activated PBMCs from donor FA (Figure 13C), and SEB-activated PBMCs from donor FB (Figure 13D). Figure 13A shows similar EC50 values ​​for all antibody derivatives shown, but the 24H9 derivatives 38A6 and 38B2 exhibit higher maximal binding signals. Same representation as in Figure 13B. Figure 13B shows lower EC50 values ​​and higher maximal binding signals compared to the 24H9 derivatives 38A6 and 38B2. Figure 13C shows similar EC50 values ​​for all antibody derivatives shown, but the 24H9 derivatives 31B1 and 33C4 exhibit lower EC50 values ​​and higher maximal binding signals compared to the 24H9 derivatives 38A6 and 38B2. Figure 13D shows similar EC50 values ​​for all antibody derivatives shown, but the 24H9 derivatives 38A6 and 38B2 exhibit higher maximal binding signals. Figures 13C and 13D. 23H9 derivatives 31B1 and 33C4 show higher maximum binding signals and lower EC50 values ​​compared to 24H9 derivatives 38A6 and 38B2, as determined using PBMCs from two different donors. [Figure 13-2] Continuation of Figure 13-1. [Figure 14] 14A and 14B depict the KD values ​​obtained for binding of antibodies 38B2, 38A6, 33C4, and 31B1 to hPD-1-his (FIG. 14A) and to hPD-1-Fc (FIG. 14B), as determined by SPR. [Figure 15]Figure 15A illustrates the ability of antibodies 31B1 and 38B2 to block intercellular hPD-1-hPD-L1 interaction. The EC50 values ​​averaged from two experiments for 31B1 and 38B2 were 0.168 and 0.154 μg / ml, respectively. The fold induction of luminescence was also averaged from two experiments. No significant difference was observed between 31B1 and 38B2. Both 31B1 and 38B2 were superior to Control B (nivolumab, sold under the trade name Opdivo) and similar to Control M (pembrolizumab, sold under the trade name Keytruda). Figure 15A. Experiment 1. Figure 15B. Experiment 2. [Figure 16] Figure 16 illustrates the effect of anti-PD-1 antibodies 31B1 and 38B2 on IL-2 cytokine production in SEB-activated PBMCs derived from donors EA (Figure 16A) and FA (Figure 16B). Cytokine IL-2 secretion was examined in SEB-activated human PBMC cells derived from donors EA and FA. The EC50 values ​​for 38B2, 31B1, ctM, and ctB were 0.101, 0.297, 0.210, and 0.548 nM (PBMCs from donor EA). Maximum IL-2 secretion by cells treated with 38B2, 31B1, ctM, or ctB was 8.05, 7.73, 7.21, and 6.79 ng / ml, respectively. 38B2 was more potent than 31B1 and two control antibodies; 31B1 was more potent than control B and comparable to control M. [Figure 17-1] Figure 17A depicts the dose-dependent binding of antibodies m3A7 and R3A9 to soluble mPD-1-Fc (Figure 17A), to soluble streptavidin-captured mPD-1-Fc (Figure 17B), to soluble streptavidin-captured mPD-1-his (Figure 17C), and to cells expressing mPD-1 (Figure 17C) as determined by ELISA, as well as blocking of mPD-1 to PD-L1 by antibodies m3A7 and R3A9 (Figure 17E), and blockade of intercellular mPD-1 / PD-L1 interactions as determined by Promega assay (Figure 17F). [Figure 17-2] Continued from Figure 17-1. [Figure 17-3]Continued from Figure 17-2. [Figure 18-1] Figure 18 illustrates the binding of antibodies 38B2, m2C1, m2A1, m4D1, m3A7, and R3A9 to hPD-1 (Figure 18A, labeled as in Figure 18B), mPD-1 (Figure 18B), and streptavidin-captured biotin-mPD-1 (Figure 18C, labeled as in Figure 18B) as determined by ELISA, and blocking of the mPD-1-mPD-L1 interaction (Figure 18D, labeled as in Figure 18E) and hPD-1-hPD-L1 interaction (Figure 18E) by antibodies 38B2, m2C1, m2A1, m4D1, m3A7, and R3A9 as determined by ELISA. [Figure 18-2] Continuation of Figure 18-1. [Figure 18-3] Continued from Figure 18-2. [Figure 19-1] 19A-19D show the binding of antibodies m2A1, m2C1, m4D1, R3A9, m3A7, and 38B2 to cells expressing hPD-1 or mPD-1. Cells expressing human PD-1: hPD-293 (Figure 19A, labeled as in Figure 19D) and hPD-1 Jurkat (Figure 19B, labeled as in Figure 19D). Cells expressing mouse PD-1: Figure 19C (labeled as in Figure 19D) and Figure 19D. [Figure 19-2] Continuation of Figure 19-1. [Figure 20] Figure 20A shows blocking of mPD-1 / mPD-L1 interaction and blocking of hPD-1-hPD-L1 interaction by antibodies 38B2, m4D1, and m2C1. Figure 20A. Blocking: m4D1 was as potent as m3A7 in blocking the mPD1-mPDL1 interaction. IC50 m4D1=0.5724nM. IC50 m3A7=0.5615nM. IC50 m2C1=1.127nM. Figure 20B. m4D1 blocks the hPD1 / hPDL1 interaction, but is not as potent as 38B2. IC50 38B2=0.7248nM. IC50 m4D1=2.125nM. IC50 m2C1=31.25nM. [Figure 21A]FIG. 1 depicts a schematic diagram of an exemplary PD-1 / IL-15 fusion protein. [Figure 21B] FIG. 1 depicts a schematic diagram of an exemplary PD-1 / IL-15 fusion protein. [Figure 22-1] Figure 22 depicts the binding of fusion proteins 1N-m3A7 / IL-15, 2N-m3A7 / IL-15, 1C-m3A7 / IL-15, and 2C-m3A7 / IL-15 to mPD-1 (Figure 22A) and to cells expressing mPD-1 (Figure 22B) and the blocking of mPD-L1 to mPD-1 using competitive ELISA (Figure 22C) or a Promega blocking assay (Figure 22D). [Figure 22-2] Continuation of Figure 22-1. [Figure 23-1] Figure 23C shows the effect of fusion proteins 1N-m3A7 / IL-15, 2N-m3A7 / IL-15, 1C-m3A7 / IL-15, and 2C-3A7 / IL-15 on the proliferation of CTLL2 (Figure 23A) and C57BL6 (Figure 23B). Figure 23C shows the effect of fusion proteins 1N-m3A7 / IL-15, 2N-m3A7 / IL-15, 1C-m3A7 / IL-15, and 2C-m3A7 / IL-15 on the proliferation of CTLL2 (Figure 23A) and C57BL6 (Figure 23B). Figure 23C shows the effect of fusion proteins 1N-DP47 / IL-15, 2N-DP47 / IL-15, 1C-DP47 / IL-15, and 2C-DP47 / IL-15 on the proliferation of CTLL2 (Figure 23A) and C57BL6 (Figure 23B). Figure 23D ...E shows the effect of fusion proteins 1N-DP47 / IL-15, 2N-DP47 / IL-15, 1C-DP4 [Figure 23-2] Continuation of Figure 23-1. [Figure 24] Figure 1 shows the ability of anti-PD-L1 / SD15 fusion proteins to reduce tumor growth in mice bearing PD-1-expressing tumors in the PD-1 / PD-L1-resistant Lewis lung model (LL2). [Figure 25A] FIG. 1 illustrates that, compared to C-terminal fusions, N-terminal fusion proteins are particularly useful for acting against tumor infiltrating lymphocytes (TILs) compared to peripheral T cells, due to the cis-presentation enabled by the N-terminal fusion constructs. [Figure 25B] FIG. 1 illustrates that, compared to C-terminal fusions, N-terminal fusion proteins are particularly useful for acting against tumor infiltrating lymphocytes (TILs) compared to peripheral T cells, due to the cis-presentation enabled by the N-terminal fusion constructs. [Figure 26-1] Figures 26A and 26B illustrate blocking of PD-1 / PD-L1 interactions (Figures 26A and 26C) and PD-1 / PD-L2 interactions (Figure 26B) for hPD-1 fusion proteins (Figures 26A and 26B) and mutant hPD-1 fusion proteins (containing the (38B2) / N65D / S / A mutant IL-15) (Figure 26C). [Figure 26-2] Continuation of Figure 26-1. [Figure 27-1] Figure 10 illustrates the stimulation of proliferation of M07e cells by the indicated anti-PD-L1-SD15 fusion proteins. [Figure 27-2] Continuation of Figure 27-1. [Figure 28] Figure 28 illustrates the effect of different anti-PD-L1 / IL-15 / linker / IL-15Rα sushi domain fusion proteins on tumor size. A CT26 syngeneic model was used. Single dose, IV. Anti-PD-L1 antibody: D7A8. [Figure 29-1] Figure 29 illustrates that the presence of the N65S mutation in IL-15 in the 1N-fusion protein does not affect the fusion's ability to bind to PD-1 and disrupt PD-1 interaction. Figure 29A. Binding of 1N-fusion protein to soluble hPD-1-Fc. Figure 29B. ELISA competition assay to measure PD-L1 binding to PD-1. Figure 29C. ELISA competition assay to measure PD-L2 binding to PD-1. Figure 29D. Binding of 1N-fusion protein to hPD-1-transfected HEK293 cells. Figure 29E. Promega blocking assay to determine PD-L2 binding to PD-1. Figure 29F. Promega blocking assay to determine PD-L2 binding to PD-1. A fusion protein containing antibody DP47 (non-targeting control antibody) served as a control. mut=N65S. [Figure 29-2] Continuation of Figure 29-1. [Figure 29-3] Continuation of Figure 29-2. [Figure 30-1]Figures 30A, 30B, and 30C illustrate that the presence of the N65S mutation in IL-15 in the 1N-fusion protein leads to a significant reduction in the efficacy of the fusion protein in the proliferation of human cells. Figures 30A, 30B, and 30C show stimulation of M07e cell proliferation by anti-PD-1 (38B2) / N65D / S / A mutant IL-15 fusion. A fusion protein containing a non-targeting control antibody served as a control (Figure 30B). Figure 30D shows proliferation of hPBMCs upon stimulation with the indicated fusion proteins. Figure 30E shows binding of the indicated fusion proteins to hIL2Rβ-transfected HEK30 cells. [Figure 30-2] Continuation of Figure 30-1. [Figure 30-3] Continuation of Figure 30-2. [Figure 31-1] Figures 31A and 31B illustrate that the presence of the N65S mutation in IL-15 in the 1N-fusion protein leads to a significantly reduced potency of the fusion protein in the proliferation of mouse cells and a greater dose response in the proliferation of CD8+ T cells than CD4+ T cells. Figures 31A and 31B show the proliferation of mouse CTLL2 cells (Figure 31A) and mouse BALB / c spleen cells (Figure 31B) upon stimulation with the indicated fusion proteins. Figures 31C and 31D show the CD8+ and CD4+ T cell populations in mouse BALB / c spleen cells (Figure 31C) and hPBMCs (Figure 31D) upon stimulation with the indicated fusion proteins. [Figure 31-2] Continuation of Figure 31-1. [Figure 31-3] Continuation of Figure 31-2. [Figure 32-1]Figures 32A, 32B, 32F, 32G, 32I, and 32K illustrate that 1N-fusion proteins containing IL-15 mutations showed potent anti-tumor activity in vivo in anti-PD-1 antibody-resistant mouse models. Figures 32A, 32B, 32F, 32G, 32I, and 32K. Tumor volume after treatment with Keytruda or the indicated 1N-fusion proteins. mut = N65S mutation. ct = DP47 = non-targeting control antibody. Keytruda = pembrolizumab (formerly lambrolizumab, trade name Keytruda). Figure 32C. Tumor growth in individual mice (N65S-38B2 / IL-15, 12 mg / kg treatment). Figures 32D, 32H, and 32J. % change in body weight. Figure 32E. Mouse survival at various dose levels. Figures 32A, 32B, 32C, 32D, 32E, 32F, 32G, 32H, 32I: Dosing was intraperitoneal once per week for 3 weeks for the 1N-fusion protein and intraperitoneal twice per week for 3 weeks for the control. Figures 32J and 32K: Dosing was intraperitoneal twice per week for 3 weeks. [Figure 32-2] Continuation of Figure 32-1. [Figure 32-3] Continuation of Figure 32-2. [Figure 32-4] Continuation of Figure 32-3. [Figure 32-5] Continuation of Figure 32-4. [Figure 32-6] Continuation of Figure 32-5. [Figure 33] FIG. 1 shows that N65S-1N-38B2 / IL-15 and N65D-1N-38B2 / IL-15 fusion proteins prevent rechallenge in hPDL1-CT26 tumor-bearing mice. [Figure 34A] Figure 34A shows that 1N-fusion proteins containing IL-15 mutations showed improved anti-tumor activity in hPD1 / PDL1 mice bearing hPDL1-CT26 tumors compared to the combination of a non-targeting fusion protein and an anti-PD-1 antibody. Figure 34B shows tumor volume after treatment with the indicated proteins. [Figure 34B]Figure 34B. Mouse survival after treatment with the indicated proteins. [Figure 34C] Figure 34C shows that 1N-fusion proteins containing IL-15 mutations showed improved anti-tumor activity in hPD1 / PDL1 mice bearing hPDL1-CT26 tumors compared to the combination of a non-targeting fusion protein and an anti-PD-1 antibody. Figure 34D. Percent change in body weight after treatment with the indicated proteins. [Figure 35A] Figure 35A shows that additional administration of anti-PD-1 antibody does not lead to a further increase in the anti-tumor activity of 1N-fusion proteins. Figure 35A shows tumor volumes after treatment with the indicated proteins. [Figure 35B] Figure 35B shows that additional administration of anti-PD-1 antibody does not lead to a further increase in the anti-tumor activity of 1N-fusion proteins. Mouse survival after treatment with the indicated proteins. [Figure 35C] Figure 35C shows that additional administration of anti-PD-1 antibody does not lead to a further increase in the anti-tumor activity of 1N-fusion proteins. Figure 35C. Percent change in body weight after treatment with the indicated proteins. [Figure 36A] FIG. 36 illustrates that N65S-1N-38B2 / IL-15 exhibited a similar serum half-life profile as antibody 38B2 in both IV (FIG. 36A) and IP (FIG. 36B) injections with a dose response up to 120 hours post-treatment. [Figure 36B] FIG. 36 illustrates that N65S-1N-38B2 / IL-15 exhibited a similar serum half-life profile as antibody 38B2 in both IV (FIG. 36A) and IP (FIG. 36B) injections with a dose response up to 120 hours post-treatment. [Figure 37A]Figure 37 illustrates the results of a mechanism of action study in hPDL1 / PD1 transgenic BALB / c mice bearing hPDL1-CT26 tumors. Figure 37A. 0.5 x 106 hPDL1 / CT26 cells were inoculated subcutaneously into the right lower flank of hPD1 / PDL1 transgenic BALB / c mice. When tumors reached approximately 175 mm in size, mice were intravenously injected with: (1) N65S-1N-38B2 / IL-15 (0.06 μmol / kg and 0.035 μmol / kg, respectively), (2) N65S-1N-DP47 / IL-15 (non-targeting control, 0.035 μmol / kg), (3) antibody 38B2 (0.035 μmol / kg), and (4) a combination of antibody 38B2 (0.035 μmol / kg) and N65S-1N-38B2 / IL-15 (0.035 μmol / kg). [Figure 37B] Figure 37B illustrates the results of a mechanism of action study in hPDL1 / PD1 transgenic BALB / c mice bearing hPDL1-CT26 tumors. Blood and draining lymph nodes (DLNs) from all groups were collected for flow cytometry. Tumors were collected for TIL analysis. [Figure 37C] Figure 37C illustrates the results of a mechanism of action study in hPDL1 / PD1 transgenic BALB / c mice bearing hPDL1-CT26 tumors. Figure 37C. CD8 / CD4 ratios 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-targeting fusion protein N65S-1N-DP47 / IL-15, or (4) a combination of 38B2 and N65S-1N-38B2 / IL-15. [Figure 37D]Figure 37D illustrates the results of a mechanism of action study in hPDL1 / PD1 transgenic BALB / c mice bearing hPDL1-CT26 tumors. (Figure 37D) Analysis of CD8 and CD4 subtypes in tumors treated with antibody (1) 38B2, (2) the PD1-targeted fusion protein N65S-1N-38B2 / IL-15, (3) the non-targeting fusion protein N65S-1N-DP47 / IL-15, or (4) a combination of 38B2 and N65S-1N-38B2 / IL-15. [Figure 38] Figure 1 illustrates that the N65S-1N-m3A7 / IL-15(surrogate) fusion protein demonstrated significant anti-tumor efficacy in multiple mouse syngeneic tumor models. EMT6 data shown as an illustrative example. DETAILED DESCRIPTION OF THE INVENTION

[0070] The present disclosure provides fusion proteins comprising an anti-PD-1 antibody or antigen-binding fragment thereof linked to an IL-15 polypeptide, which in turn is linked to an IL-15 receptor alpha (IL-15Rα) polypeptide comprising an IL-15Rα sushi domain. Further provided herein are antibodies and antigen-binding fragments thereof that specifically bind to PD-1, as well as fusion proteins comprising such anti-PD-1 antibodies and PD-1-binding fragments thereof.

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

[0072] Fusion proteins containing a stimulatory domain are disclosed herein. As used herein, a "stimulatory domain" is a domain that promotes an immune response. The 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 receptors that respond 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, which is a heterocomplex of two or more ligands covalently linked to each other.

[0073] In embodiments, the stimulatory domain comprises a sequence or domain that enhances 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 an 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 an SD15 domain highlighted by any one of SEQ ID NOs: 177-180.

[0078] As demonstrated herein, provided herein are fusion proteins 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, which fusion proteins provide increased immune cell activity compared to two separate molecules that separately provide the aforementioned functions. Specifically, experiments disclosed herein demonstrate that a fusion protein containing both a PD-1-binding domain that blocks the binding of PD-1 to PD-L1 and a stimulatory domain, an IL-15 or derivative thereof, and an IL-15Rα polypeptide containing an IL-15Rα sushi domain or derivative thereof, promoted increased proliferation, Th1 cytokine release, and molecules associated with NK and T cell killing activity compared to providing the domains in separate molecules.

[0079] In certain embodiments, the fusion protein comprises a stimulatory domain disclosed herein covalently linked to an anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein via a flexible linker. In some embodiments, provided herein are fusion proteins in which a stimulatory domain disclosed herein is fused directly to an anti-PD-1 antibody or antigen-binding fragment thereof 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, two covalently linked polypeptides are fused directly to each other, i.e., without any additional polypeptide sequence between the first and second polypeptides. Thus, in some embodiments, the N-terminus of the first polypeptide is fused directly to the C-terminus of the second polypeptide, or vice versa. In other embodiments, two covalently linked polypeptides 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 two or more polypeptides fused to each other.

[0081] IL-15 is a 14- to 15-kD cytokine with structural similarity to IL-2. IL-15 is also known as MGC9721. Various cell types constitutively produce IL-15 mRNA, including monocytes, macrophages, DCs, keratocytes, epithelial skin cells, fibroblasts, various epithelial cells, bone marrow stromal cells, and neurons. 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 bind to the same hematopoietin subunit and share many biological activities. IL-15 regulates T and NK cell activation and proliferation and promotes CD8 + The number of memory cells is affected by the balance between IL-15 and IL-2. In embodiments, the IL-15 or IL-15 derivatives disclosed herein have 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. The IL-15 may be mammalian IL-15, preferably primate IL-15, and more preferably human IL-15. The amino acid sequence of human IL-15 (Accession No. 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, an IL-15 or IL-15 derivative sequence may contain one or more amino acid substitutions. In some embodiments, the amino acid substitutions are located at positions 1, 4, 8, 30, 45, 61, 64, 65, and / or 108 of IL-15 (residue numbering refers to the mature form of the IL-15 protein). 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 in IL-15 are N1D, N4D, D8N, D30N, D61N, E64Q, L45A, N65S / A / D / K, and / or Q108S / E. In some embodiments, the amino acid substitutions in IL-15 are L45A, N65S / A / D / K, and / or Q108S. In embodiments, a fusion protein disclosed herein comprises an IL-15 polypeptide comprising one of the following amino acid substitutions: N65S, N65A, or N65D. In embodiments, a fusion protein disclosed herein comprises 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 No. NP_002180) is provided in SEQ ID NO: 213. The IL-15 receptor, i.e., the IL-15 receptor complex, binds IL-15 specifically with high affinity and consists of a unique interleukin-15 receptor α subunit, IL-2 / IL-15Rβ and the common γ chain / IL-2Rγ subunit. IL-15Rα binds mitogen-activated macrophages, NK cells, and CD4 + and CD8 +Human IL-15Rα is expressed by T cells. Human IL-15Rα consists of seven exons, and alternative mRNA splicing can result in eight molecular IL-15Rα isoforms with distinct 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 the domain beginning with the first cysteine ​​residue (C1) after the signal peptide of IL-15Rα and ending with the fourth cysteine ​​residue (C4) after the signal peptide. The sushi domain, which corresponds 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 human IL-15Rα isoform 1 precursor is provided in SEQ ID NO: 213 (this sequence includes the signal sequence). The sushi domain amino acid sequence of human IL-15Rα is provided in SEQ ID NO: 214.

[0086] The term "IL-15Rα sushi domain derivative" or "IL-15Rα sushi domain variant" 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 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 IL-15Rα sushi domain. In some such derivatives, naturally occurring amino acids may be replaced with chemically modified amino acids 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 (IL-15Rα residue numbering includes the signal peptide, see e.g., SEQ ID NO: 213). In some embodiments, the amino acid substitution in the IL-15Rα sushi domain is N60A (i.e., the 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). 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 proteins disclosed herein comprise a stimulatory 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 stimulatory 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 stimulatory domains exemplified herein are fused directly to an anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein.

[0089] In one embodiment, an "N-terminal fusion protein" is provided in which a stimulatory domain is linked, either directly or via a linker (e.g., via one or more peptides), 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).

[0090] N-terminal fusion proteins include, but are not limited to, the following (exemplary) fusion proteins (components 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 containing an 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 containing an 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-15 or a derivative thereof)-(IL-15Rα polypeptide containing an IL-15Rα sushi domain or a derivative thereof)-(heavy chain of an anti-PD-1 antibody or an antigen-binding fragment thereof) (6) (IL-15 or a derivative thereof)-(IL-15Rα polypeptide containing an IL-15Rα sushi domain or a derivative thereof)-(light chain of an anti-PD-1 antibody or an antigen-binding fragment thereof)

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

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

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

[0094] In one embodiment, a fusion protein is provided in which a stimulatory domain is covalently linked to the N-terminus of both a heavy chain of an anti-PD-1 antibody, or antigen-binding fragment thereof. 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 a stimulatory domain is linked, either directly or via a linker (e.g., via one or more peptides), 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 disclosed herein).

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

[0097] The C-terminal fusion protein may contain more than one stimulatory domain.

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

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

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

[0101] FIG. 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 can also cause toxicity because IL-15 shares two receptor subunits with IL-2. However, when presented in cis, N-terminal IL-15 fusion proteins demonstrate reduced toxicity compared to C-terminal fusion proteins, as they can selectively bind to tumor-infiltrating lymphocytes (TILs) while exhibiting lower binding to peripheral T cells. When presented in cis, N-terminal fusion proteins can simultaneously bind to IL-15Rβγ and PD-1 while reducing IL-15Rβ / γ binding, allowing N-terminal fusion proteins to selectively bind to TILs and promote TIL proliferation. In contrast, C-terminal fusion proteins may bind equally to both peripheral T cells and TILs and exhibit higher toxicity than N-terminal fusion proteins (Figure 25). The 1 N-terminal fusion protein exhibits reduced toxicity compared to an N-terminal fusion protein comprising two stimulatory domains covalently linked to an anti-PD-1 antibody disclosed herein (2 N-terminal fusion protein). The mutated N-terminal fusion protein exhibits further reduced toxicity due to lower binding to peripheral T cells.

[0103] In one aspect, the present disclosure provides a method for manufacturing a method of a cellular membrane comprising: (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 connecting the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide; The present invention relates to a fusion protein comprising:

[0104] The anti-PD-1 antibody or antigen-binding fragment thereof, portion of the fusion protein, can be any that specifically binds to PD-1, including those that contain the corresponding variable regions or CDRs provided in Figure 1 or otherwise described herein. In embodiments, the fusion protein comprises an antibody or antigen-binding fragment thereof, wherein 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 is 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 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 CDR3L is 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.

[0105] In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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 antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:24 or a sequence 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 SEQ ID NO:98 or a sequence 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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 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 NO: 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 antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:24 or a sequence 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 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 NOs: 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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), X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L is X5X6SX7X8X9X 10 (SEQ ID NO: 63), 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 MQGX 11 X 12 WPYT (SEQ ID NO: 64), X 11 is A, T or S; X 12 is H or R.

[0110] In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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 antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:20 or a sequence 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 SEQ ID NO:51 or a sequence 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 embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein 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: 17, 18 and 19, respectively; The sequences of CDR1L, CDR2L and CDR3L 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, respectively.

[0113] In a further embodiment, the fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof, wherein: the heavy chain variable region comprises SEQ ID NO:20 or a sequence 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 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 NOs: 44, 54, 57 or 61.

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

[0115] Provided herein are fusion proteins comprising a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 174-184, 187-190, 193-199, 206-211, and 224-243. Provided herein are fusion proteins comprising a light chain comprising a sequence selected from the group consisting of SEQ ID NOs: 170, 173, 186, 192, and 205.

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

[0117] Provided herein are fusion proteins comprising a light chain and one or more heavy chains listed in Table 27.

[0118] In embodiments, 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:

[0119] In embodiments, 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:

[0120] In embodiments, 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:

[0121] In embodiments, 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:

[0122] In embodiments, 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:

[0123] In embodiments, 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:

[0124] Antibodies and antigen-binding fragments thereof The term antibody is used herein in the 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, mutated antibodies, and grafted antibodies (grafted antibodies), bispecific antibodies, specific antibody portions (e.g., domain antibodies), and any antigen-binding portion thereof that competes with the intact antibody for specific binding, its antigen-binding portion (e.g., paratope, CDR), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site, so long as it exhibits the desired biological activity and specificity. Thus, an antibody is an immunoglobulin molecule or a fragment or derivative thereof, including any polypeptide that contains an antigen-binding site and is capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The disclosed antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).

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

[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 polypeptides is approximately 23,000 daltons, while the molecular weight of the heavy chain polypeptides varies between 53,000 and 70,000 daltons. The four chains are typically connected by disulfide bonds in a "Y" configuration.

[0127] When immunoglobulins are produced by either hybridomas, B cells, or genetically engineered host cells, the two heavy chains (HC) and two light chains (LC) of an immunoglobulin molecule are covalently bound to each other, and the terminal portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. Both 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 regions of hypervariability 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 amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0128] The variable region enables an antibody to recognize and specifically bind to an epitope located on an antigen. The variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. The antigen-binding site of an antibody is composed of the VL domain and the VH domain or a subset of 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 fixation, etc. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody.

[0129] As used herein, the term "complementarity-determining region" (CDR) refers to the portion of an antibody variable domain that is (usually) involved in antigen binding. Each variable region has three noncontiguous CDRs known as CDR1, CDR2, and CDR3. The CDRs are separated by structurally conserved regions called framework regions (FR-1, -2, -3, and -4), which form a "core" β-sheet structure that displays these loops on the surface of the variable domain. The six CDRs present in each antigen-binding domain are short, noncontiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The length and composition of the CDR sequences are highly variable, particularly in CDR3. The remainder of the amino acids located in the antigen-binding domain or "framework" region show less inter-molecular variability. The antigen-binding domain formed by the arranged CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. Each CDR may, for example, comprise amino acid residues from a CDR as defined by Kabat (i.e., approximately 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 may also comprise amino acid residues from a "hypervariable loop" (i.e., approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (Chothia & Leskowicz, 1996). J. Mol. Biol. 901 (1987)). In some examples, the CDRs may contain amino acids from both the CDR regions defined according to Kabat and the hypervariable loops.Kabat numbering does not always correspond to the linear numbering of amino acid residues due to shortening or insertion of structural components, whether framework or CDR, of the basic variable domain structure. The correct Kabat numbering of residues for a given antibody or antigen-binding fragment thereof can be determined by aligning homologous residues in the sequence of the antibody or antigen-binding fragment thereof 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, 55-77 (2003)).

[0130] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" can be a fragment comprising a Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs such as shark and camel antibodies), ScFv, maxibody, minibody, nanobody, intrabody, diabody, triabody, tetrabody, v-NAR, and bis-scFv, or a polypeptide containing at least a certain portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0131] The antibody may be of any class, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any particular class but encompasses any immunoglobulin molecule containing an antigen recognition site of the required specificity, other modified configurations, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Modified versions of each of these classes and isotypes are known to those of skill in the art and, accordingly, are 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 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, which refers 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 that includes the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0136] A single-chain Fv or scFv antibody fragment comprises or consists essentially of the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables 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 comprising or essentially consisting 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 comprising 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 comprising, or consisting essentially of, a VH domain.

[0139] In some embodiments of the aspects described herein, the anti-PD-1 antibody fragment is a F(ab')2 fragment, which comprises a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.

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

[0141] Those skilled in the art can use a variety of techniques that have been developed and are available for producing antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies. However, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody fragment of choice is a single-chain Fv fragment (scFv). See, e.g., WO 93 / 16185. Alternatively, these fragments can also be produced 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 directly recovered 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 contains complementary regions that bind to PD-1.

[0143] Contemplated antibodies or antigen-binding fragments can have any type of constant region, 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. An antibody or antigen-binding fragment thereof can contain sequences from more than one class or isotype.

[0144] Anti-PD-1 antibodies and their PD-1-binding fragments This 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 PD-1 or fragments thereof coupled to, for example, 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 for 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 can interfere with, inhibit, or reduce PD-1 biological activity, including downstream events mediated by PD-1. The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein can exhibit any one or more of the following functions: (a) blocking binding to PD-1 and 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 potent binding and inhibitory activity and are useful for therapeutic and diagnostic uses.

[0146] In one aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to PD-1. 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 disclosure provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to PD-1, and 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 comprising 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 is the sequence RX 13 X 14 X15 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 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 CDR3L is 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 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 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 embodiments, the antibody or antigen-binding fragment thereof is a heavy chain variable region comprising SEQ ID NO:24 or a sequence 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 Includes:

[0151] In embodiments, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof 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 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 NO: 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.

[0152] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the following: a heavy chain variable region comprising SEQ ID NO:24 or a sequence 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: 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 NOs: 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. Includes:

[0153] 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: 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), X1 is Q or E; X2 is H or Y; X3 is Y or N; X4 is T or N; The sequence of CDR2L is X5X6SX7X8X9X 10 (SEQ ID NO: 63), 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 MQGX 11 X 12 WPYT (SEQ ID NO: 64), X 11 is A, T or S; X 12 is H or R.

[0154] 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: 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.

[0155] In embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the following: a heavy chain variable region comprising SEQ ID NO:20 or a sequence 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. Includes:

[0156] In embodiments, the CDRs of the anti-PD-1 antibody or antigen-binding fragment thereof comprise the following sequences: the sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOs: 17, 18 and 19, respectively; The sequences of CDR1L, CDR2L and CDR3L 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, respectively.

[0157] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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 a 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 selected from the group consisting of SEQ ID NOs: 28, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, and 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 a light chain variable region amino acid sequence selected from the group consisting of: 6, 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 disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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 disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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 a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 51, 54, 57, and 61.

[0160] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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:51.

[0161] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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:54.

[0162] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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:57.

[0163] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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:61.

[0164] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:138.

[0165] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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 a light chain variable region amino acid sequence 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 disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:98.

[0167] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:69.

[0168] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:72.

[0169] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:76.

[0170] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:79.

[0171] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:83.

[0172] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:85.

[0173] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:87.

[0174] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:90.

[0175] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:91.

[0176] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:93.

[0177] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:95.

[0178] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:101.

[0179] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:105.

[0180] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:107.

[0181] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:109.

[0182] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:112.

[0183] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:114.

[0184] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:115.

[0185] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:117.

[0186] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:120.

[0187] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:121.

[0188] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:123.

[0189] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:126.

[0190] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:128.

[0191] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:131.

[0192] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: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 of SEQ ID NO:134.

[0193] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein 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: 142, 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 a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 150, 153, 156, 160, 164, and 167.

[0194] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 143; The sequence of CDR2L comprises the sequence SEQ ID NO: 144; The sequence of the CDR3L comprises the sequence SEQ ID NO:145.

[0195] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 147; The sequence of CDR2L comprises the sequence SEQ ID NO: 148; The sequence of the CDR3L comprises the sequence SEQ ID NO:149.

[0196] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 151; The sequence of CDR2L comprises the sequence SEQ ID NO: 148; The sequence of the CDR3L comprises the sequence SEQ ID NO:152.

[0197] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 154; The sequence of CDR2L comprises the sequence SEQ ID NO: 148; The sequence of the CDR3L comprises the sequence SEQ ID NO:155.

[0198] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 157; The sequence of CDR2L comprises the sequence SEQ ID NO: 158; The sequence of the CDR3L comprises the sequence SEQ ID NO:159.

[0199] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 161; The sequence of CDR2L comprises the sequence SEQ ID NO: 162; The sequence of the CDR3L comprises the sequence SEQ ID NO:163.

[0200] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment comprises 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 SEQ ID NO: 139; The sequence of CDR2H comprises the sequence SEQ ID NO: 140; The sequence of CDR3H comprises the sequence SEQ ID NO: 141; The sequence of CDR1L comprises the sequence SEQ ID NO: 157; The sequence of CDR2L comprises the sequence SEQ ID NO: 165; The sequence of the CDR3L comprises the sequence SEQ ID NO:166.

[0201] In addition to the above description, Figure 1 shows the heavy and light chain variable regions and associated CDRs of the antibodies disclosed herein, as well as the accompanying sequence listing, which is incorporated by reference in its entirety. As shown in the Examples below, light chain shuffling of anti-PD-1 antibodies led to the identification of antibody variants that confer substantial improvements in PD-1 binding affinity. Analysis of the variants revealed certain CDR positions where amino acids remained relatively invariant among the light chain-shuffled antibodies, as well as other CDR positions where variations could be introduced without abolishing PD-1 binding.

[0202] The anti-PD-1 antibodies and antigen-binding fragments thereof disclosed herein, and 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 antibodies or antigen-binding fragments thereof disclosed herein (or fusion proteins comprising the anti-PD-1 antibodies or antigen-binding fragments) have been altered by amino acid substitution, deletion, insertion, and / or addition. The amino acid substitutions may be conservative or non-conservative. The present disclosure also includes anti-PD-1 antibodies and antigen-binding fragments thereof (and fusion proteins comprising the anti-PD-1 antibodies or antigen-binding fragments) that are derived from the amino acid sequences disclosed herein and in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). In certain embodiments, an anti-PD-1 antibody or binding fragment thereof (or a fusion protein comprising the anti-PD-1 antibody or antigen-binding fragment) comprises one or more CDRs, or one or more variable domains, having 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 Figure 1.

[0203] Also provided herein are variable heavy and variable light chain sequences and pairings thereof that are similar, but not identical, to the variable heavy and variable light chains and pairings thereof 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 antigen-binding fragment thereof utilizes a framework described in Table 25.

[0204] Also provided herein are chimeric antigen receptors (CARs) that comprise one, two, three, four, five, or six CDRs of the anti-PD1 antibodies and antigen-binding fragments disclosed herein. Also provided herein are CARs that comprise six CDRs of any one of the anti-PD1 antibodies and antigen-binding fragments disclosed herein.

[0205] Disclosed is an immune cell that expresses a CAR that comprises one, two, three, four, five or six CDRs of the anti-PD1 antibody and antigen-binding fragment disclosed herein.Disclosed is an immune cell that expresses a CAR that comprises six CDRs of any one of the anti-PD1 antibody and antigen-binding fragment 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 optimally aligned sequences, after taking into account the number of gaps required for optimal alignment and the length of each 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 protein function.

[0207] Also disclosed herein are anti-PD-1 antibodies, or antigen-binding fragments thereof, or fusion proteins comprising 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 an amino acid sequence disclosed herein. Methods and computer programs for determining sequence similarity are publicly available, including, but not limited to, 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). The Smith Waterman algorithm can also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, MD 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] Some embodiments of the aspects described herein contemplate amino acid sequence modifications of an antibody or antigen-binding fragment thereof that binds to PD-1, or a fusion protein that binds to PD-1 described herein. Amino acid sequence variants of an anti-PD-1 antibody or antigen-binding fragment thereof, or fusion protein, are prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the anti-PD-1 antibody or antigen-binding fragment thereof, 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. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., binding specificity, inhibition of biological activity.

[0209] Amino acid substitutions can be made in some cases by selecting substitutions that are not significantly different in their effect on (a) the structure of the peptide backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk (conservative amino acid substitution variants). These variants have at least one amino acid residue in an antibody or antigen-binding fragment thereof or fusion protein replaced by a different residue with similar side chain properties. Amino acids can be grouped according to the similarity of their side chain properties (see Lehninger, BIOCHEMISTRY (2nd ed.), 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 polarity: 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, a non-limiting example of a conservative amino acid substitution is one that replaces a non-polar amino acid with another non-polar amino acid.

[0211] Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) Hydrophobicity: Ala(A), Val(V), Leu(L), Ile(I), Met(M); (2) Neutral hydrophilicity: 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. Non-limiting examples of substitutions include: 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] Amino- and / or carboxyl-terminal fusions ranging in length from one to 100 or more residues, as well as amino acid sequence insertions, which may include intrasequence insertions of single or multiple amino acid residues, are further contemplated. Examples of terminal fusions include an antibody or antigen-binding fragment thereof with an N-terminal methionyl residue, or an antibody or antigen-binding fragment thereof fused to a cytotoxic polypeptide (or a fusion protein comprising such an antibody or antigen-binding fragment thereof). Other examples of terminal fusions of an antibody or antigen-binding fragment thereof include the fusion to the N- or C-terminus of the antibody or antigen-binding fragment thereof (or a fusion protein comprising such an antibody or antigen-binding fragment thereof) of an enzyme or polypeptide that increases the serum half-life of the antibody or antigen-binding fragment thereof, such as biotin.

[0214] Any cysteine ​​residue not involved in maintaining the proper conformation of an antibody or antigen-binding fragment thereof that binds PD-1 can also be substituted, e.g., with serine or alanine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

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

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

[0217] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof may contain modifications including, but not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization, by known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Chemical modification processes are known in the art and include, but are 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 can include anti-PD-1 antibodies and antigen-binding fragments thereof whose binding characteristics have been altered by direct mutation, affinity maturation, phage display, or chain shuffling. Affinity and specificity are altered by mutating the CDRs and screening for CDRs with the desired characteristics. Mutagenesis methods are known to those skilled in the art.

[0219] antibody binding Also provided herein are anti-PD-1 antibodies and antigen-binding fragments thereof, as well as fusion proteins comprising an anti-PD-1 antibody or antigen-binding fragment thereof that binds to the same epitope on PD-1 as 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. In one embodiment, the anti-PD-1 antibodies or antigen-binding fragments thereof do not exhibit 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 generates a soluble form of PD-1. In one aspect, the present disclosure provides anti-PD-1 antibodies or antigen-binding fragments thereof that bind to all isoforms of PD-1. In some embodiments, the provided anti-PD-1 antibodies and antigen-binding fragments thereof bind to mammalian PD-1.

[0221] As used herein, "binding" of an antibody or antigen-binding fragment thereof, or a fusion protein comprising the antibody or antigen-binding fragment thereof, to PD-1, an epitope on PD-1, or, in certain embodiments described below, a specific residue on PD-1, includes the selective interaction of the antibody or antigen-binding fragment thereof 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 antibodies or antigen-binding fragments thereof described herein bind to PD-1 in an amount 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 -10In other embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof described herein bind to PD-1 with an equilibrium constant of dissociation (KD) of 10 mol / L. -2 ~10 -9 mol / 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 in mol / l.

[0223] As used herein, the "affinity" of an antigen, expressed by KD, with an antigen-binding protein is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein, such as an antibody or antibody fragment thereof. D The value of K is inversely proportional to the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can also be expressed as 1 / K D The association constant (K A The affinity can be determined by a person 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 antigen-binding fragment thereof, such as an anti-PD-1 antibody or 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 competitive assays or by the epitope identification / characterization techniques described herein or equivalents known in the art.

[0225] The term "epitope" as used herein refers to the specific target to which an antibody binds. Epitopes are formed both by a continuous stretch of amino acids (continuous epitopes) and by a three-dimensional arrangement of amino acid residues that are present only when the target protein is folded into a specific conformation (discontinuous epitopes). Generally, epitopes contain at least three amino acids, at least four, at least five, or about 7-10 amino acids.

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

[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 antigen-binding fragment thereof binds to PD-1 and inhibits PD-1 activity and / or PD-1 binding 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, competing, cross-blocking, 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 competing or cross-blocking antibody to human PD-1 prevents the binding of an antibody 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 an anti-PD-1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, five of the six CDRs of an anti-PD-1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, six of the six CDRs of an anti-PD-1 antibody or antigen-binding fragment thereof contact the antigen.

[0229] The terms "selective" and "selectivity," as used herein, 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 particular region, target, or peptide; typically, a region or epitope in PD-1, as opposed to one or more other biomolecules, including other PD-1 family members.

[0230] In one aspect, anti-PD-1 antibodies and antigen-binding fragments thereof are provided that specifically bind to at least a portion of the binding site on PD-1, thereby blocking PD-1 interaction with one or more PD-1 ligands, including, but 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 comprises an Fc domain composed of a first and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains comprising the heavy chain domain 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, or an IgG4 Fc domain. In further specific 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 antibodies or antigen-binding fragments thereof comprises a modification that promotes 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 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] Knob-into-hole engineering is a "protrusion-into-cavity" strategy that operates to engineer the interface between a first and second polypeptide for hetero-oligomerization. A "protrusion" (i.e., knob) is constructed by replacing a small amino acid side chain from the interface of a first polypeptide with a larger one. A compensatory "cavity" (i.e., hole) of the same or smaller size as the protrusion is optionally created on the interface of a second polypeptide by replacing a large amino acid side chain with a smaller one. In certain embodiments, an amino acid residue in the CH3 domain of a first Fc subunit is replaced with an amino acid residue having a larger side chain volume, thereby generating a knob in the CH3 domain of the first Fc subunit that can be positioned into a hole present in the CH3 domain of a second Fc subunit, generated by replacing an 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 knobs and holes can be made by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0235] In fusion protein embodiments in which only one heavy chain is linked to 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 a heavy chain lacking the fusion). In embodiments, an amino acid residue in the CH3 domain (CH3-1) of one heavy chain contains an amino acid substitution that replaces the amino acid with an amino acid residue having a larger side chain volume, thereby creating a "knob" in the CH3 domain that can be positioned into a "hole" present in the CH3 domain (CH3-2) of the other heavy chain created 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 of the Fc domain (the "knob" subunit) may comprise 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 of the Fc domain (the "hole" subunit) may comprise one or more substitutions from the following: Y349C, T350V, T366L, T366S, L368A, K392L, T394W, Y407V, Y407T.

[0236] In some embodiments, the amino acid substitution is: (1) First heavy chain CH3 domain: S354C, T366W; second heavy chain CH3 domain: 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: Y407T; (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) is selected from.

[0237] Provided herein is an antibody or antigen-binding fragment thereof (or a fusion protein comprising such an antibody or antigen-binding fragment thereof) that binds to PD-1, wherein 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] Provided herein is an antibody or antigen-binding fragment thereof (or a fusion protein comprising such an antibody or antigen-binding fragment thereof) that binds to PD-1, wherein 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, an Fc domain in accordance with the present disclosure exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain, hi certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to an unengineered Fc domain.

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

[0241] The linker amino acid sequences described herein can be of sufficient length to ensure that the fusion protein forms the appropriate secondary and tertiary structure. The length of the linker amino acid 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 comprises near-neutral amino acids selected from the group including Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L) and Gln (Q), most preferably from the group including Gly (G), Asn (N) and Ser (S). Preferably, the linker sequence is glycine- and serine-rich, and in some embodiments, the linker contains only serine and glycine residues.

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

[0244] A non-limiting example of a linker is a portion of an amino acid sequence: 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) Examples include:

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

[0246] In embodiments, the linker connecting the IL-15 polypeptide to the anti-PD-1 antibody heavy chain is GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 216) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 244) Includes:

[0247] In embodiments, the heavy and light chains of an anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein may be connected into a single polypeptide chain ("single-chain Fv" or "scFv") using a third linker that allows the VR and VL domains to associate and form the 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 to the IL-15Rα sushi polypeptide or IL-15Rα sushi derivative by a linker.

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

[0250] In one embodiment, the IL-15Rα sushi or IL-15Rα sushi derivative is covalently linked to the anti-PD-1 antibody or 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 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 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 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 antigen-binding fragment thereof by a second linker. In some embodiments, the amino acid sequences of the first and second linkers are identical. In other embodiments, the amino acid sequences of the first and second linkers are different.

[0252] Conjugates 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 moieties, and therapeutic moieties.

[0253] Blocking moieties may contain moieties of sufficient steric bulk and / or charge to result in reduced glycosylation, for example, by blocking the ability of sucrose to glycosylate an antibody or antigen-binding fragment thereof. Preferred blocking moieties include cysteine ​​adducts such as cysteine, 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 its apparent molecular weight. 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, soluble in water, heat stable, inert to many chemicals, non-hydrolyzable, and non-toxic, making it a favorable 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, imaging agents, radioisotope moieties, radio-opaque moieties, and the like, such as detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Examples of fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other light-emitting molecules (e.g., luminal). Fluorophores can be environmentally sensitive, such that their fluorescence changes when located in close proximity to one or more residues in the modified protein that undergo a conformational change upon binding to a substrate (e.g., a dansyl probe). Exemplary radiolabels include small molecules containing one or more atoms with low sensitivity nuclei (e.g., C, N, H, I, I, Tc, K, Fe, Ga, Ga, In, etc.).

[0255] The diagnostic moiety comprises a detectable moiety suitable for revealing the existence of a disease or disorder. Typically, the diagnostic moiety allows for determining the presence, absence or level of molecules, such as target peptides, proteins (singular or plural), associated with a disease or disorder. Such diagnostics are also suitable for predicting and / or diagnosing a 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 agents. Functional moieties can also have one or more of the above functions. Exemplary therapeutic moieties include antibiotics, a second anti-PD-1 antibody, or an antibody against another antigen, such as a tumor-specific antigen, an autoimmune tissue antigen, or a virally 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, autoimmune disease, or chronic viral infection. Exemplary therapeutic moieties can also include cytotoxins, radioactive agents, cytokines, interferons, target or reporter moieties, enzymes, toxins, peptides, or therapeutic agents at any position along the molecule, as long as they are capable of binding to their target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In certain embodiments, an antibody is conjugated to an agent specific for tumor cells or virally infected cells.

[0257] For example, salvage receptor binding epitopes, such as those described in U.S. Patent No. 5,739,277, can be attached to antibodies or antigen-binding fragments thereof (particularly antibody fragments) to increase the half-life of the antibodies or antigen-binding fragments described herein. The term "salvage receptor binding epitope" can refer to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for 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, as well as 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 any length, either ribonucleotides or deoxyribonucleotides, including, but 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. Nucleic acids encoding the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be, for example, DNA, cDNA, RNA, synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any of these polynucleotides, alone or in combination.

[0259] The term "vector" refers to a vehicle containing a nucleic acid molecule that can transport the nucleic acid molecule into a cell. "Vector" includes, but is not limited to, a viral vector, a plasmid, an RNA vector, or a linear or circular DNA or RNA molecule, which may be composed of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid. In some embodiments, the vectors used are capable of autonomous replication (episomal vectors) and / or expression (expression vectors) of the nucleic acids to which they are linked. Several suitable vectors are known to those of skill 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 typically produced by recombinant expression. Optionally, nucleic acids encoding the light and heavy chain variable regions linked to constant regions can be inserted into the same expression vector. Alternatively, optionally, the nucleic acids encoding the light and heavy chain variable regions linked to 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 a eukaryotic host cell. Typically, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences and for the collection and purification of cross-reactive antibodies after the vector has been incorporated into the appropriate host.

[0261] Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance or neomycin-resistance) to permit detection of cells transformed with the desired DNA sequences.

[0262] The host used to express the anti-PD-1 antibodies, antigen-binding fragments thereof, or fusion proteins disclosed herein can be a prokaryotic or eukaryotic host. Examples of suitable hosts include yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or bacterial or eukaryotic hosts, including host cells of mammalian, insect, avian, or yeast origin. 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 can be Escherichia coli, bacilli such as Bacillus subtilus, and other enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.

[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 contain expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like, as needed. Common promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in methanol, maltose, and galactose utilization, among others.

[0265] Cultured mammalian cells can also be used as host cells to 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) 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 contain expression control sequences such as replication origins, promoters, and enhancers, as well as 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 papilloma virus, cytomegalovirus, etc.

[0266] The anti-PD-1 antibodies, antigen-binding fragments thereof, 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 antibody heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, whole antibodies disclosed herein, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, etc. (See generally, Scopes, Protein Purification (Springer-Verlag, NY, (1982)). For pharmaceutical uses, substantially pure immunoglobulins of at least about 90-95% homogeneity are preferred, with 98-99% or more homogeneity being 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 murine antibodies or fusion molecules are known in the art.

[0268] Methods for modulating PD-1 activity In one aspect, the disclosure provides methods of using the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins 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 antibodies, antigen-binding fragments thereof, or fusion proteins disrupt 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" refers to the ability to cause an overall decrease 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, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more compared to untreated controls. Immunosuppression is mediated by immune inhibitory receptors expressed on the surface of immune cells and their interaction with their ligands. Methods for measuring T cell activity are known in the art. As a non-limiting example, T cell tolerance can be induced by contacting T cells with a recall antigen, anti-CD3, in the absence of costimulation and / or ionomycin. For example, levels of IL-27, LDH-A, RAB10, and / or ZAP70 (both intracellular and secreted) can be monitored to determine, for example, the degree of T cell tolerance (with levels of IL-2, interferon-γ, and TNF correlating 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 a key role in the immune system in regulating 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 may compromise this regulatory mechanism, driving immunosuppression. Blocking PD-1 with antagonists, including monoclonal antibodies, has been investigated in the treatment of cancer and chronic viral infections. PD-1's ability 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, causing these T cells to 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 immune response, the therapeutic agent disclosed herein that antagonizes PD-1 signal transduction can be administered to treat diseases involving PD-1-mediated immunosuppression.

[0273] Immunomodulation is a useful therapeutic approach for treating various diseases and disorders. One approach to immunomodulation is to intervene at one or more immune checkpoints by using regulators of immune activation, which play an important role 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 that present non-self antigens can avoid immune attack by secreting cytokines or ligands that activate immune checkpoints. Therefore, 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 certain tissues.

[0274] In one aspect, the disclosure provides anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins that are 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. As used herein, the term "mammal" includes, but is not limited to, humans, laboratory animals, domestic pets, and livestock. Preferably, the mammal is a human. As used herein, a "therapeutically effective amount" refers to an amount of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein that, when administered to a mammal, is effective to produce a desired therapeutic effect.

[0276] By "subject" is meant a mammal, including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, sheep, or cat, etc. Individuals and patients are also subjects herein.

[0277] The terms "treat," "treated," "treating," or "treatment," as used herein, refer to therapeutic treatment whose purpose is to slow (reduce) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; delaying or slowing the onset of the progression of a condition, disorder, or disease; improvement of one or more symptoms of the condition, disorder, or disease state; and remission (whether partial or complete). Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival if not receiving treatment. Terms such as "prevent," "prevention," and the like refer to acting before the onset of an overt disease or disorder in order to prevent the disease or disorder from developing, minimize the extent of the disease or disorder, or slow the course of development.

[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 myopathy, 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 a subject suffering from a chronic viral infection. 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, the anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be used to treat a chronic viral infection by administering a therapeutically effective dose 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 embodiment, 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 cancer, 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 therapeutically effective amounts to a subject suffering from cancer or a viral infection.

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

[0283] Combination therapy The anti-PD-1 antibodies, antigen-binding fragments thereof, and fusion proteins disclosed herein can be advantageously 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, immunosuppressants, and anti-infective agents. The administration of the anti-PD-1 antibodies, antigen-binding fragments thereof, or fusion proteins and the additional therapeutic agents can be simultaneous, sequential, or intermittent. The administration of the anti-PD-1 antibodies, antigen-binding fragments thereof, or fusion proteins and the additional therapeutic agents can be separate or as a mixture. Furthermore, the methods of treatment provided herein may involve treatment in combination with one or more therapies, including, but not limited to, 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 thereof, and fusion proteins of the present disclosure can be synergistically combined with one or more anti-cancer drugs or therapies used to treat cancer, including, but not limited to, renal cell carcinoma, colorectal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, 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, an antibody against PD-L1 together with a second antibody against PD-1 (e.g., nivolumab), a 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 ligand antagonist (e.g., CD-28, 2B4, LY108, LAIR).1, 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 as set forth in US 7,087,411 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 beta (TGF3) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), agonists for costimulatory receptors (e.g., agonists for glucocorticoid-inducible TNFR-associated protein), 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, cobicistat, tenofovir), Antibodies against ribovirin, rilpivirine, and corticosteroids), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., bacillus Calmette-Guérin, cancer vaccines), adjuvants to enhance 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), cytotoxins, chemotherapeutic agents (e.g., For example, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, 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-DM4Anti-DS6-DM4 ADCs and anti-DS6-DM4 ADCs), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), nutritional supplements such as antioxidants or any palliative care for treating cancer, radiation therapy, and / or antibodies against Fc receptors on immune cells for the treatment of autoimmune diseases.

[0285] Administration method Therapeutic compositions comprising any of the anti-PD-1 antibodies, antigen-binding fragments thereof, 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, placement, or implantation. The compositions described herein can be administered to a subject in need thereof subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intracranially, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.

[0286] In certain embodiments, the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein is administered to a mammal by intravenous infusion, i.e., introduction of the anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein into the vein of the mammal over a certain period of time, which in certain embodiments 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 disclosure involves sequentially administering to a subject multiple doses of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure. As used herein, "sequentially administering" means that each dose of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure is administered to a subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, days, weeks, or months). The disclosure includes methods comprising sequentially administering to a patient a single initial dose of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure, followed by one or more second doses of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein of the present disclosure, and optionally, followed by one or more third doses of an 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, a compound or composition is administered to a subject every day, 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 a compound or composition are administered to a subject every day, every few days, every third day, once a week, once every two weeks, or once a month. In some embodiments, a compound or composition is administered for 2, 3, 5, 7, 14, 21, or 28 days. In certain embodiments, a 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 more.

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

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

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

[0292] Therapeutic compositions comprising an anti-PD-1 antibody, or antigen-binding fragment thereof, or fusion protein thereof, can be formulated with one or more pharmaceutically acceptable excipients, which can be pharmaceutically acceptable materials, compositions, or vehicles such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, magnesium talc, calcium stearate or zinc stearate or stearic acid), solvents or encapsulating materials involved in carrying or transporting the therapeutic compound for administration to a subject, bulking agents, salts, surfactants, and / or preservatives. Some examples of materials that can serve as pharmaceutically acceptable excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, 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; buffers; water; isotonic saline; pH buffer solutions; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0293] Bulking agents as referred to herein are described as compounds that are added to increase the mass of pharmaceutical compositions and contribute to the physical structure of the formulation in lyophilized form. Examples of bulking agents include, but are not limited to, suitable mannitol, glycine, polyethylene glycol and sorbitol.

[0294] The therapeutic composition may optionally contain a surfactant. The use of a surfactant may reduce aggregation of the reconstituted protein and / or reduce particle formation in the reconstituted formulation. Examples of suitable surfactants that may be used according to 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 glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamide protease. Examples of suitable surfactants include pyro-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl tauate or disodium methyl oleyl tauate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic, PF68, etc.).

[0295] Preservatives can optionally be used in the therapeutic compositions described herein.Suitable preservatives for use in the formulations provided herein include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (the alkyl group is long-chain compound, alkylbenzyl-dimethylammonium chloride mixture) and benzethonium chloride.Other types of preservatives include aromatic alcohols such as phenol, 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 varying concentrations of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein. For example, the compositions may contain 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 of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein. The compositions may also contain approximately 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 of an anti-PD-1 antibody, antigen-binding fragment thereof, or fusion protein. In some embodiments, the therapeutic composition can be lyophilized and provided in a composition for reconstitution prior to administration.

[0297] Diagnostic Use The anti-PD-1 antibodies, antigen-binding fragments, 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, or fusion proteins disclosed herein can be used in assays to detect diseases or disorders such as cancer, autoimmune diseases, or chronic viral infections. An exemplary diagnostic assay for PD-1 may include, for example, contacting a sample obtained from a patient with an anti-PD-1 antibody, antigen-binding fragment, or fusion protein of the present disclosure, which is labeled with a detectable label or reporter molecule, or used as a capture ligand to selectively isolate PD-1 from the patient sample, or alternatively, used in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be an isotope such as 3H, C, 32P, 35S, or 251; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme 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, administration 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 The kit may comprise one or more of the antigen-binding fragment or fusion protein, a carrier composition, an administration device, and a combination therapeutic agent.The kit may further comprise a device for facilitating delivery, such as a syringe for injection, or a tool for facilitating delivery of the therapeutic composition to a subject in need thereof.Any of the kits provided herein can be contained in a container, pack, or dispenser together with instructions for administration.

[0299] All other mentioned patents and applications are incorporated herein by reference in their entirety. Furthermore, to the extent that a definition or use of a term in a reference incorporated herein by reference 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 reference 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 vary. 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. Furthermore, it should be understood that the present disclosure herein includes all possible combinations of such specific configurations. For example, if a specific configuration is disclosed in the context of a particular aspect or embodiment of the present disclosure or a particular claim herein, that configuration can also be used, to the extent possible, in combination with and / or in the context of other specific aspects and embodiments of the present disclosure, and in the disclosure generally.

[0301] When a method including two or more defined steps is referred to herein, the defined steps may be performed in the order listed, or in any order, or simultaneously (unless the context excludes that possibility), and the method may include one or more other steps performed before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes that possibility).

[0302] In order to facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given, which should not be read as limiting or defining the entire scope of the present disclosure. [Example]

[0303] Identification of anti-PD-1 antibodies from the Distributed Bio SuperHuman 2.0 Inc. library hPD-1-Fc (human IgV domain fused to the N-terminus of human IgG1Fc; present as a dimer in solution) or hPD-1-his (human IgV domain of PD-1 connected to the N-terminus of a six-histidine peptide; present as a monomer in solution) was immobilized on immunotubes coated with anti-his or anti-hFc antibodies. Biotin-labeled hPD-1-his was captured by streptavidin-conjugated magnetic beads. The phage library was blocked with 3% PBS milk and added to the antigen-coated tube or mixed with the antigen-coated beads and incubated at room temperature for 0.5–1 h to allow phage to bind to the immobilized antigen. Unbound phage was washed away, while bound phage was eluted with triethylamine (pH > 11). Neutralized phage was used to infect log-phase E. coli, which was grown overnight at 30°C. The recovered bacteria were used to amplify phage for the next round of panning.

[0304] The heavy and light chain variable domains of selected scFv antibodies isolated from the phage library were amplified by PCR and inserted into mammalian expression vectors containing modified human IgG1 constant domains with effector functions removed. Full-length IgG antibodies were expressed in HEK293 cells and purified by Protein A. Purified anti-19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 (SEQ ID NOS: 1-48, see 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 with SEB.

[0305] To identify higher affinity antibodies, two light chain shuffling libraries were constructed by combining the heavy chains of lead antibodies 23H9 or 24H9 with the light chain mixture from R1 panning. Higher affinity and / or more stable variants were identified by panning under more restrictive conditions. These matured 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 antibodies for further studies. Light chain shuffling using the 23H9 heavy chain yielded antibodies 31B1, 33C4, 33G8, and 34C1 (SEQ ID NOs: 49-65, see Table 25). Light chain shuffling using the heavy chain of 24H9 yielded 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).

[0306] Figure 1 shows a sequence alignment of exemplary antibodies generated herein. Table 25 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-PD-1 antibodies according to the Kabat CDR definitions. The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples provided below. [Example]

[0307] Antibodies isolated from a phage display library bind to hPD-1 The ability of anti-hPD1 antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 (isolated from HEK293 cells) to bind to hPD1 was assessed using a dose-response binding ELISA with the indicated antibody as the primary antibody. To this end, high-binding (Immulon 2HB) plates were directly coated with human PD-1-Fc (R&D, catalog no. 1086-PD-050). Biotin-labeled hPD-1-his (Sino Biologicals, catalog no. 10377-H08H) was captured with streptavidin (Jackson 016-000-114) or anti-his antibody (R&D, catalog no. MAB050-100) coated onto the plate one day prior. Serial dilutions of antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 were added to the antigen-coated plates and incubated for 1-2 hours at room temperature. After washing the plates three times with PBST (0.05%), anti-hFab-HRP antibody (Jackson 109-036-097) diluted 1 / 5000 to 1 / 10,000 was added to the plates and incubated for 1 hour at room temperature. Color was developed by adding a 1:1 dilution of TMB substrates A and B (Fisher, catalog number 5120-0050) followed by neutralization with 1N H2SO4. OD450 readings were taken using a TECAN plate reader (Table 1). The mean ± SD of OD450 readings was plotted against the log antibody concentration using GraphPad Prism 8. EC50 values ​​were calculated by plotting log(agonist) vs. response (3-parameter) or log(agonist) vs. response-variable slope (4-parameter) (Figure 2). Data points in Figure 2 represent the mean ± SD of duplicate determinations. When bound to human PD-1-Fc, all six antibodies had similar EC 50 and O.D. 450 (10 nM) values, but when bound to PD-1-his, different EC 50 and O.D. 450(10 nM) values ​​were observed (Table 1). Antibodies 23A8, 23H9, and 24H9 showed superior 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] [Example]

[0309] Antibodies isolated from a phage display library block the hPD-1 / hPD-L1 interaction. To examine the ability of anti-hPD1 antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 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 no. 156-B7-100) or PD-L2-Fc (R&D, catalog no. 1224_PL), respectively, and blocked with 3% PBS milk. Serial dilutions of antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 were mixed with 0.5 μg / ml (final concentration) of biotin-labeled hPD-1-Fc and incubated at room temperature for 1 hour. The mixture was transferred to a PD-L1-Fc or PD-L2-Fc-coated plate and incubated for an additional 1-2 hours at room temperature. After washing the plate three times with PBST (0.05%), streptavidin-HRP (Thermofisher, catalog no. 21140) diluted 1 / 5000 to 1 / 10,000 was added to the plate and incubated at room temperature for approximately 1 hour. Color was developed by adding a 1:1 dilution of TMB substrates A and B, followed by neutralization with 1N H2SO4. OD450 was read using a TECAN plate reader (Table 2). The mean ± SD of the OD readings was plotted against the log antibody concentration using GraphPad Prism 8. EC50 values ​​were calculated by analyzing the log (agonist) vs. response (3-parameter) or log (agonist) vs. response slope (4-parameter). Data points for duplicate determinations are means ± SD (Figure 3). All six antibodies were found to block hPD-1-Fc binding to human PD-L1 with different IC50 values ​​(Table 2 and Figure 3). All six antibodies have very similar affinities for hPD-Fc. However, antibodies 23A8, 23H9, and 24H9 showed superior 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% 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 (R-phycoerythrin-conjugated goat anti-human IgG: Jackson ImmunoResearch catalog number 109-116-098). Antibody binding to hPD-1 was determined using a Guava EasyCyte™ HT sampling flow cytometer (EMD Millipore). Data points in Figure 4 represent the mean ± SD of duplicate determinations. 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 PBMCs: PBMCs were isolated from LeukoPak (enriched leukopheresis 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 2 × 10 in 12-well plates containing IMDM (Gibco, Life Technologies) supplemented with 10% FBS (GE Healthcare HyClone).6 Cells were cultured at 1000xygen / mL and activated with 1 μg / mL SEB (Staphylococcal enterotoxin B, Sigma) for 3–4 days. Activated PBMCs were first incubated with the indicated anti-PD-1 antibodies, followed by incubation with a combination of PE-anti-human IgG1-Fc (R-phycoerythrin-conjugated goat anti-human IgG; 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). Antibody binding to PD-1 expressed on the cells was assessed using a Guava EasyCyte™ HT sampling flow cytometer (EMD Millipore). Data points were generated by calculating the mean ± SD of duplicate determinations (Table 3 and Figure 4). Antibodies 19B6, 23A8, 23H9, and 24H9 exhibited the highest MFI and lowest EC50 for binding to PBMCs among the antibodies tested and were selected for further characterization (Table 3 and Figure 4).

[0313] [Table 3] [Example]

[0314] Antibodies isolated from a phage display library disrupt intracellular PD-1 / PD-L1 interactions To assess the ability of 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 blocking 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, PD-1 / PD-L1 interaction inhibits TCR-mediated luminescence. When PD-1 / PD-L1 interaction is disrupted, TCR activation induces luminescence (by activating the NFAT pathway), which can be detected by adding Bio-Glo™ reagent and quantitating using a luminometer.

[0315] 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 antibodies and PD-1 effector cells were added. After 6 hours of incubation at 37°C, Bio-Glo™ reagent was added, and luminescence (recorded as relative light units, RLU) was measured using a TECAN plate reader. Fold induction was calculated by RLU (induced minus background) / RLU (no antibody control minus background). Data points are presented as the mean ± SD of duplicate determinations (Figure 5). Antibodies 19B6, 23A8, 23H9, and 24H9 blocked the hPD-1 / hPD-L1 interaction with 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 library of light chains, respectively. Antibodies 31B1, 33C4, 33G8, and 34C1 (SEQ ID NOs: 49-65, see 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 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 abilities of anti-hPD1 antibody 23H9 and its derivatives 31B1, 33C4, 33G8, and 34C1 to bind to hPD1 and block the hPD-1 / hPD-L1 interaction were determined using the protocols described in Examples 2 and 3, respectively. The parent antibody 23H9 and its derivatives showed no significant differences in binding to hPD-1-Fc and biotin-hPD-1-his captured by streptavidin (Table 5 and Figure 6A). However, all derivatives were more potent than its parent 23H9 in binding to hPD-1-his captured by anti-his and in blocking the hPD-1-F / hPD-L1-Fc interaction (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 abilities of anti-hPD1 antibody 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 were determined using the protocols described in Examples 2 and 3, respectively. The parent 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 parent 24H9 in binding to hPD-1-his captured by anti-his and in blocking the hPD-1-Fc-hPD-L1-Fc interaction (Figure 7).

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

[0322] [Table 6] [Example]

[0323] Binding profiles of 23H9 and 24H9 derivatives indicate that these antibodies bind to different epitopes Dose-response binding assays using 23H9, 31B1, 33C4, 24H9, 38A6, and 38B2 were 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 in binding to PD-1-expressing cells than their parent antibodies (Table 7). Furthermore, the 23H9 and 24H9 derivatives exhibited distinct binding profiles, indicating that they bind to different epitopes (Figure 8).

[0324] [Table 7] [Example]

[0325] Antibodies 23H9, 24H9, and their derivatives disrupt the intercellular PD-1 / PD-L1 interaction 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 parent 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 greatest fold induction among the six antibodies tested (Table 8 and Figure 9).

[0326] [Table 8] [Example]

[0327] Effect of anti-PD-1 antibody on cytokine production in SEB-activated PBMCs The effect of anti-PD-1 antibodies (38A6, 38B2, 31B1, and 33C4) on cytokine production in SEB-activated PBMCs from donor FA was assessed by measuring the amount of cytokines released by PBMCs in the culture medium. PBMCs were isolated from LeukoPak (a concentrated leukopheresis containing a high concentration 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 × 10 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 SEB (Staphylococcal enterotoxin B, Sigma) for 2 days. Supernatants were collected for IL-2 measurement using a Duoset ELISA kit (R&D Systems) according to the manufacturer's instructions (Figure 10A).

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

[0329] IL-2 concentrations increased in a dose-dependent manner in the presence of all antibodies (Figures 10A and 10B). IL-2 concentrations increased in the presence of the 23H9 derivative 31B1 and the 24H9 derivative 38B2, which were found to be more potent than their parent 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 antibodies to either hPD-1-Fc or hPD-1-his were measured by surface plasmon resonance (SPR) using a Biacore T200. Human PD-1-Fc or PD-1-his proteins were immobilized onto a Series S CM5 sensor chip at pH 5 using standard amine coupling chemistry. Antibodies 23H9 and 23H9 derivatives 31B1 and 33C4, as well as 24H9 and 24H9 derivatives 38A6, 38B2, 32D11, 39G8, 38A10, and 38A11, were injected over the immobilized surface at concentrations ranging from 1.5 to 100 nM at 30 μl / min using 1X HBSEP as the running buffer. The contact time (association phase) was 3 min. The dissociation time was 6 to 10 min. Regeneration was performed after each binding cycle using an injection of 20 mM HCl at a flow rate of 30 μl / min for 30 s. 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 parent 23H9 in their binding to hPD-1-Fc (Table 10). All 24H9 derivatives were found to be more potent than the parent antibody 24H9 in their binding to hPD-1-his and hPD-1-Fc.

[0332] [Table 10] [Example]

[0333] Thermal stability measurements 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 was greater than 96% with endotoxin levels lower than 1.0 EU / mg. Differential scanning calorimetry (DSC) was used to determine the stability of the four antibodies. DSC analysis was performed using a Malven DSC system. Protein samples were first diluted to 1 mg / mL with formulation buffer before analysis. 400 μL of each formulation buffer was added to a 96-well plate as a reference, followed by 400 μL of protein sample. Samples were heated from 10°C to 100°C at a heating rate of 90°C / h in a capillary DSC system. The DSC results (Tm onset and Tm values) were analyzed using the supplier's software. The lowest thermal 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 percentages of all antibodies were greater than 96% with endotoxin levels lower than 1.0 EU / mg (Table 11). 33C4 and 38B2 were found to be the most thermostable of all antibodies tested (Table 11).

[0334] [Table 11] [Example]

[0335] Selected 23H9 and 24H9 derivatives bind to hPD-1 and block hPD-1 / hPD-L1 and hPD-1 / hPD-L2 interactions Binding and blocking ELISAs were performed using antibodies 31B1, 33C4, 38A6, and 38B2, which were transiently isolated from CHOk cells, using the protocols outlined in Examples 2 and 3, respectively. Comparison of the antibodies using binding ELISAs revealed no significant differences among the derivatives in binding to hPD-1-Fc, biotin-labeled 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 superior to both controls (Table 12 and Figures 12A-C).

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

[0337] [Table 12]

[0338] [Table 13] [Example]

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

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

[0341] [Table 14] [Example]

[0342] Selected 23H9 and 24H9 derivatives bind to hPD-1 with high affinity The binding of 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, among the two derivatives of 23H9, 31B1 was found to be more potent than 33C4 in binding to both PD-1 Fc and PD-1 his (Table 15). Among the two derivatives of 24H9, 38B2 was found to be slightly more potent than 38A6 in binding to hPD-1 Fc and showed no significant difference in 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 intracellular PD-1 / PD-L1 interaction To evaluate the ability of 31B1 and 38B2 to disrupt the binding of cells expressing PD-1 to cells expressing PD-L1, a Promega PD-1 / PD-L1 blockade 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 similar to Control M (pembrolizumab, sold under the trade name Keytruda) (Table 16).

[0345] [Table 16] [Example]

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

[0347] [Table 17] [Example]

[0348] Isolation of human and mouse cross-reactive antibodies from the Distributed Bio SuperHuman 2.0 Inc. library PD-1 antibodies that bind to both mouse and human PD-1 were 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 N-terminally to six histidines; exists as a monomer in solution).

[0349] Derivatives of anti-mPD-1 antibody R3A9 (SEQ ID NOs: 139-146) were generated by light pair shuffling using the VH chain of R3A9. R3A9 and its derivatives (see, e.g., SEQ ID NOs: 147-167) were selected for further development. The antibodies were cloned and expressed by WuXi Biologics using Wuxi's mammalian expression vector and CHO knockout cell line. 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 selected antibodies generated according to the methods of this example are detailed in the Examples set forth below. Figure 1 and Table 25 provide the amino acid sequences of the heavy and light chain variable regions and CDRs of selected anti-mPD-1 antibodies (Kabat numbering). [Example]

[0351] Anti-mPD-1 antibody R3A9 and its derivatives bind to mPD-1 and block the mPD-1 / mPD-L1 interaction Dose-response binding and blocking ELISAs were performed using R3A9 and its derivatives using protocols as outlined in Examples 2 and 3. Binding of antibodies to cells expressing mPD-1 was performed as described in Example 4. Blockade of intercellular PD-1 / PD-L1 interactions 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 soluble, cell-expressed mPD-1 and blocking the mPD-1 / mPD-L1 interaction (Table 18).

[0353] [Table 18] [Example]

[0354] R3A9 derivatives m2C1, m2A1, m4D1, and m3A7 bind to human and mouse PD-1 Dose-response binding and blocking ELISAs were performed on the antibodies using the protocols outlined in Examples 2 and 3, respectively. The experiments showed that all R3A9 derivatives potently bound to both hPD-1 and mPD-1 (Figures 18A-C) and blocked 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] [Example]

[0356] R3A9 binds to cells expressing human or mouse PD-1 R3A9 and its R3A9 derivatives m2A1, m2C1, m4D1, m3A7 were tested for binding to PBMC cells using the protocol outlined in Example 4. Additionally, Biacore analysis was performed on all 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 on cells (Table 21 and Figure 19). Based on cell binding (Table 20) and Biacore analysis (Table 20), m4D1 was found to exhibit improved binding properties 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 Blockade Bioassay Comparing m4D1 with 38B2 and m3A7 A Promega blocking bioassay was performed to compare the potency of m4D1, m3A7, and 38B2 to disrupt the intercellular PD-1 / PD-L1 interaction using the protocol outlined in Example 4. 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 and three-fold less potent than 38B2 in mPD-1 / mPD-L1 blocking assays (Figure 20). [Example]

[0362] Fusion proteins containing anti-mPD1 or anti-hPD-1 antibodies, IL-15, and IL-15Rα sushi domains An anti-mPD-1 fusion antibody, including antibody m3A7 (referred to as m3A7 / IL-15), was engineered by appending the IL-15Rα sushi domain and an IL-15 polypeptide to either the C-terminus or N-terminus of the antibody. The fusion of the IL-15Rα sushi domain with part of the hinge region, linker 1, and IL-15 is referred to as "SD15." For an example of an SD15 domain, see SEQ ID NO: 174 in Table 25, with 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 a 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 a 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, the light chain was that of a conventional antibody. To eliminate complement binding and fixation and Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC), amino acid substitutions L234A, L235A, P329G (LALA-PG) were introduced into both CH2 domains.

[0368] Additionally, for heterodimeric fusions, mutations were introduced into the constant region of the heavy chain, with amino acid substitutions 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 antibody was transiently expressed in CHOk1 cells. After purification using Protein A and SEC-HPLC, a monomer with at least 95% purity was obtained. Four corresponding versions of the anti-hPD-1 / IL-15 fusion antibody containing antibody 38B2 (referred to herein as 38B2-hSD15 or 38B2 / IL-15)—1C-38B2-hSD15, 2C-38B2-hSD15, 1N-38B2-hSD15, and 2N-38B2-hSD15—were produced using the same procedure. Three corresponding versions of 1N_38B2-SD15, 65A_1N_38B2-hSD15, 65D_1N_38B2-hSd15, and 65S_1N_38B2-hSD15, which have an amino acid substitution at position 65 of the IL-15 sequence, were produced 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, and 2N-DP47 / IL-15, were generated following the same procedure. DP47 was used as a non-targeting control antibody. A schematic diagram showing 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 abilities of 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 were evaluated according to the protocols described in Examples 2 and 3, respectively.

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

[0373] [Table 22] [Example]

[0374] Effect of mPD-1 / IL-15 fusion molecule on IL-2-dependent cell proliferation of murine CTLL2 lymphocytes and C57BL6 spleen cells CTLL2 culture and proliferation assay: The murine 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 with ConA, Corning). The following fusion proteins were tested in this example: 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 antibodies or fusion proteins were added to 96-well plates, and cells were then seeded at 2.5 × 10 cells 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).

[0376] Mouse splenocyte isolation and proliferation assay: C57BL / 6 mouse spleens were homogenized in IMDM medium using a 70 μm cell strainer. 1× RBC lysis buffer (eBioscience) was added to the splenocytes, allowed to stand for 3 minutes with occasional shaking, and then diluted with 5 volumes of PBS buffer. After washing with PBS buffer, splenocyte cell suspensions were immediately prepared for proliferation assays in complete IMDM medium supplemented with 10% heat-inactivated FBS. The following fusion proteins were tested in this example: 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 fusion proteins and controls were added to 96-well plates, and cells were then seeded at 1 × 105 per well. Cells were cultured at 37°C for 5–7 days and harvested for flow cytometry analysis. Cells were washed and stained with the fixable viability dye Fluor 780 (eBioscience) for 30 minutes at 4°C. After washing, cells were fixed and permeabilized with Foxp3 transcription factor staining buffer set (eBioscience), followed by staining with CD3 (145-2C11)-PE, CD4 (RM4-5)-APC, CD8α-(53-6.7)-PE Cy7, and Ki67 (SolA15)-FITC from eBioscience. Proliferating 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 murine lymphocyte CTLL2 (Fig. 23A) and C57BL6 splenocyte proliferation (Fig. 23B). Analysis of T cell populations revealed that only the percentage of CD8+ T cell population was increased, while CD4 +It was shown that the population percentage of T cells remained unaffected (Figure 23C). Of the four constructs tested, 1N-m3A7 / IL-15 showed the least stimulatory effect on IL-2-dependent murine lymphocyte and splenocyte proliferation and T cell proliferation, while 2C-m3A7 / IL-15 showed the most stimulatory effect on cell proliferation and T cell proliferation. [Example]

[0379] Efficacy study of mPD-1 / IL-15 fusion molecule in the PD-1 / PD-L1 Lewis lung cancer model To determine whether the orientation or number of IL-15 / IL-15Rα sushi domains 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 performed 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] C57BL / 6 strain mice, 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 in DMEM medium supplemented with 10% fetal bovine serum. Cells were harvested in the logarithmic growth phase, counted, and then inoculated into tumor tissue. The fusion protein used in this example was produced by WuXi Biologics and exhibited a purity of at least 95% and contained more than 95% monomer.

[0381] The right hind flank region of each mouse was inoculated with LL / 2 tumor cells (3 × 10) in 0.1 ml of PBS for tumor development. 5The randomization date and the first administration date were designated as day 0. The mean tumor size was approximately 100 mm 3 The randomization process began when the mice reached the age of 18. The mice were randomly assigned to the different study groups. Randomization was performed using a multitasking method (StudyDirector™ software, version 3.1.399.19) randomized block design 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 injected intravenously into mice once a week for 3 weeks. After randomization, mice were weighed twice a week. Tumor volume was measured twice a week in two dimensions using calipers, and volume was expressed in mm using the formula: V = (L × W × W) / 2, where V represents tumor volume, L represents tumor length (the longest tumor dimension), and W represents tumor width (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 antitumor activity of the fusion proteins, 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 mean tumor volume (or body weight) of the treatment and control groups, respectively, for the fusion protein and control.

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

[0385] Taken together, the data from Examples 25 and 26 suggest that N-terminal fusion antibodies further reduce toxicity compared to C-terminal fusion antibodies due to lower stimulation of peripheral T cells. The data further indicate that N-terminal fusion molecules are particularly effective in stimulating tumor-infiltrating lymphocytes (TILs) compared to peripheral T cells due to cis presentation; reduced IL-2Rβγ binding in N-terminal fusion molecules allows PD-1 / IL-15 to selectively bind to TILs, thus reducing their toxicity. Finally, the data indicate that 1N-fusion molecules are superior to 2N-fusion molecules.

[0386] [Table 23] [Example]

[0387] The hPD-1 / IL-15 fusion protein retained its PD-1 / PD-L1 inhibitory function Promega blocking bioassays were 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 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] 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 from cell line 5637.

[0389] Serial dilutions of antibodies or fusion proteins were added to a 96-well plate, and the cells were then plated at 2.5 x 10 per well in IMDM medium supplemented with 10% heat-inactivated FBS. 4 The cells were incubated at 37°C for 3 to 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, however the 1N fusion showed reduced ability to stimulate compared to the 1C, 2C or 2N constructs (FIG. 27). [Example]

[0391] Efficacy study of hPD-L1 / SD15 fusion molecule in CT26 isogenic model To determine whether the orientation or number of IL-15 / IL-15Rα sushi domains has any effect on the ability of the fusion molecules to inhibit tumor growth, in vivo efficacy studies were conducted in the CT26 isogenic model using 1N-SD15-D7A8, 2N-SD15-D7A8, 1C-SD15-D7A8, and 2C-SD15-D7A8. CT26 isogenic model mice were treated with the fusion proteins and observed for tumor development / reduction. CT26 is a murine colon carcinoma line.

[0392] Tumor volume was measured twice weekly in two dimensions using calipers, and volume was expressed in mm using the formula: V = (L × W × W) / 2, where V represents tumor volume, L represents tumor length (longest tumor dimension), and W represents tumor width (longest tumor dimension perpendicular to L). 3 The doses were expressed as . Dosing and tumor and body weight measurements were performed in a Laminar Flow Cabinet. Body weight and tumor volume were measured by 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 IL-15 amino acid substitutions in the 1N-fusion protein did not affect the PD-1 / PD-L1 inhibitory function of the fusion protein. Promega blocking bioassays (Figure 26C, Figure 29E, and Figure 29F) and ELISA competition assays (Figures 29B and 29C) showed that the N65S or N65D amino acid substitutions in the IL-15-containing 1N-fusion proteins did not affect the ability of the fusion proteins to effectively disrupt PD-1 / PD-L1 or PD-1 / PD-L2 interactions.

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

[0396] 1N-fusion proteins containing IL-15 amino acid substitutions showed weak IL-15 stimulation in vitro The ability of 1N-fusion proteins 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), hPBMCs (Figure 30D), an IL-2 / IL-15-dependent mouse lymphocyte cell line (CTLL2, Figure 31A), and mouse splenocytes (Figure 31B) proliferated slower than in the presence of wild-type 1N-IL-15 / 38B2. Binding of N65S-1N- / 38B2 to hIL-2Rβ-transfected HEK293 cells was reduced (Figure 30E).

[0397] Furthermore, both 1N-IL-15 / 38B2 and mut-1N-IL-15 / 38B2 significantly increased CD4 expression in both mouse spleens (Figure 31C) and hPBMCs (Figure 31D). +CD8 rather than T cells + It induced a better dose response in T cell proliferation. [Example]

[0398] IL-15 1N-fusion proteins containing amino acid substitutions showed potent antitumor activity in vivo in anti-PD-1 antibody-resistant mouse models 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. Tumor size was approximately 100 mm. 3 When the mice reached 100 mg / kg, they were injected (intraperitoneally) with fusion antibodies (once a week) and controls (twice a week).

[0399] Both 1N-38B2 / IL-15 and N65S-1N-38B2 / IL-15 demonstrated potent antitumor activity (Figure 32A). The antitumor efficacy of N65S-1N-38B2 / IL-15 was dose-dependent (Figure 32B). N65S-1N-38B2 / IL-15 at 12 mg / kg QW for 3 weeks demonstrated tumor regression (Figures 32B and 32C). No weight loss or death was observed with N65S-1N-38B2 / IL-15 when dosed at 12 mg / kg, but significant weight loss and death were observed with 1N-38B2 / IL-15 when dosed at ≥ 1 mg / kg (Figures 32E and 32E). Non-targeting 1N-IL-15 fusions did not demonstrate any antitumor efficacy. Significant weight loss (FIG. 32D) and death were observed when dosed at ≧1 mg / kg for the non-targeted 1N-IL-15 fusion (data not shown).

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

[0401] In summary, fusion proteins containing mutant IL-15 reduced the potency and stimulation associated with IL-15, resulting in reduced toxicity and an increased therapeutic treatment window. [Example]

[0402] IL-15 1N-fusion proteins containing amino acid substitutions showed potent antitumor activity in vivo in mice rechallenged with tumor cells. The in vivo efficacy of 1N-fusion proteins containing IL-15 N65S or IL-15 N65D, respectively, was evaluated in hPD-1 / hPD-L1 transgenic mice.

[0403] 0.5×10 6 hPDL1 / CT26 cells were subcutaneously inoculated into the right lower flank of each mouse. 3 When tumor size reached 100 mg / kg, mice were injected intraperitoneally with 12 mg / kg of N65S-1N-38B2 / IL-15 or N65D-1N-38B2 / IL-15 once weekly and twice weekly for 3 weeks. Five mice (out of six) experienced complete tumor regression at approximately 25 days for N65S-1N-38B2 / IL-15 treatment and 40 days for N65D-1N-38B2 / IL-15 treatment.

[0404] On day 88, 0.5 x 10 6 hPDL1 / CT26 cells and 0.1 x 10 6 CT26 cells were inoculated subcutaneously into the left lower and upper flanks, respectively. No tumor growth was observed up to day 135 for both cell lines, indicating that the fusion protein protected mice from rechallenge with tumor cells (Figure 33). [Example]

[0405] 1N-fusion proteins containing IL-15 amino acid substitutions showed improved antitumor activity in hPD1 / PDL1 mice bearing hPDL1-CT26 tumors compared with the combination of a non-targeting fusion protein and an anti-PD-1 antibody. 0.5×106 hPDL1 / CT26 cells were subcutaneously inoculated into the right lower flank of mice. 3 When the mice reached IL-15, they were intraperitoneally injected weekly for 3 weeks with 10 mg / kg 38B2, 12 mg / kg N65S-1N-38B2 / IL-15, or a combination of 10 mg / kg 38B2 and 12 mg / kg N65S-1N-DP47 / IL-15. For combination treatment, the two test substances were injected sequentially with a 1-hour interval.

[0406] The following constructs were compared for their ability to suppress tumor growth: (1) N65S-1N-38B2 / IL-15 fusion protein; (2) non-targeting fusion N65S-1N-DP47 / IL-15; (3) combination of anti-PD-1 antibody 38B2 with a non-targeting 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 with the combination group (Figure 34A). In the N65S-1N-38B2 / IL-15 fusion group, 83% (5 / 6) of mice were tumor-free, whereas no tumor-free mice were observed in the combination group and one tumor-free mouse was observed in the anti-PD-1 antibody 38B2-only group (Figure 34B). No weight loss was observed for the 65S-1N-IL15 / 38B2 fusion group, and approximately -5% weight loss was observed for the non-targeting fusion 65S-1N-IL15 / DP47 group (Figure 34C).

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

[0409] Additional administration of anti-PD-1 antibody does not further enhance the antitumor activity of the 1N-fusion protein 0.5×10 6hPDL1 / CT26 cells were subcutaneously inoculated into the right lower flank of mice. 3 When the mice reached IL-15, they were intraperitoneally injected once weekly for 3 weeks with 10 mg / kg 38B2, 12 mg / kg N65S-1N-38B2 / IL-15, or a combination of 10 mg / kg 38B2 and 12 mg / kg N65S-1N-38B2 / IL-15. For combination treatments, the two test substances were first mixed and then injected into the mice.

[0410] The following constructs were compared for their ability to suppress tumor growth: (1) N65S-1N-38B2 / IL-15 fusion protein; (2) the combination of anti-PD-1 antibody 38B2 and N65S-1N-38B2 / IL-15 fusion protein; and (3) the 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). 83% (5 / 6) of mice were tumor-free in the N65S-1N-38B2 / IL-15 group, while no tumor-free mice were observed in the combination group and one tumor-free mouse was observed with 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 anti-PD-1 antibody. [Example]

[0413] The N65S-1N-38B2 / IL-15 fusion protein exhibited a long serum half-life in hPD1 / PDL1 transgenic mice bearing hPDL1-CT26 tumors hPD1 / PDL1 transgenic Balb / c mice were inoculated with hPDL1 / CT26 tumor cells (0.5 × 10) in 0.1 mL of PBS. 6 Animals were randomized and the mean tumor volume was approximately 83 mm 3Treatment began when the tumor reached a median age of 10 days. Mice were administered 5.1 mg / kg (0.35 μmol / kg) of 38B2 or 1 or 6 mg / kg (0.06 or 35 μmol / kg) of N65S-1N-38B2 / IL-15 intraperitoneally (IP) or intravenously (IV). After dosing, blood was collected at 0.2, 5, 2, 4, 12, 24, 48, 72, 120, and 168 hours post-injection. Purified serum was used to measure antibody concentrations by traditional ELISA using human PD1 as the captured protein and biotin-IL15 and anti-human Fab antibodies, respectively, to detect KD050 and 38B2 binding.

[0414] N65S-1N-38B2 / IL-15 exhibited a serum half-life profile similar to antibody 38B2, both IV (FIG. 36A) and IP (FIG. 36B) injection, demonstrating a dose response up to 120 hours post-treatment. [Example]

[0415] Mechanism of action study in hPDL1 / PD1 transgenic BALB / c mice bearing hPDL1-CT26 tumors 0.5×10 6 hPDL1 / CT26 cells were subcutaneously inoculated into the right lower flank of hPD1 / PDL1 transgenic BALB / c mice. Tumor size was approximately 175 mm. 3When tumor size reached 100 μg / mL, mice were intravenously injected with 0.06 or 0.35 μmol / kg of (1) N65S-1N-38B2 / IL-1, (2) N65S-1N-DP47 / IL-15 (non-targeting control), (3) antibody 38B2, and (4) a combination of antibody 38B2 and N65S-1N-38B2 / IL-15. Tumor size was measured twice weekly, and mice were terminated on day 7 post-dose (Figure 37A). Blood and draining lymph nodes (DLN) were collected from all groups for flow cytometry. Tumors were harvested for TIL analysis (Figure 37B). One-way ANOVA was used for statistical analysis. If a significant F-statistic (ratio of treatment variance to error variance) was obtained, comparisons between different groups were performed using the Games-Howell (not assuming equal variances) or Tukey (assuming equal variances) test. Comparison between vehicle and other groups, *p<0.05; **p<0.01; ***p<0.001. Error bars represent 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, analysis of the CD8 / CD4 ratio in tumors, blood, and draining lymph nodes (treated with antibody (1) 38B2, (2) the PD1-targeting fusion protein N65S-1N-38B2 / IL-15, (3) the non-targeting fusion protein N65S-1N-DP47 / IL-15, or (4) a combination of 38B2 and N65S-1N-38B2 / IL-15) revealed that N65S-1N-38B2 / IL-15 differentially expanded CD8+ T cells in tumors. + We found that 38B2 stimulated T cell proliferation in the blood but not in the draining lymph nodes. The CD8 / CD4 ratio did not change as a result of other treatments: (1) 38B2, (2) the non-targeting fusion protein N65S-1N-DP47 / IL-15, or (3) the combination of 38B2 and N65S-1N-38B2 / IL-15 (Figure 37C).

[0417] We then analyzed CD8 and CD4 subtypes in tumors treated with antibody (1) 38B2, (2) the PD1-targeting fusion protein N65S-1N-38B2 / IL-15, (3) the non-targeting fusion protein N65S-1N-DP47 / IL-15, or (4) a combination of 38B2 and N65S-1N-38B2 / IL-15. We found that effector memory CD8 T cells in tumors were significantly increased for N65S-1N-38B2 / IL-15-treated samples and slightly increased for 38B2-treated samples (Figure 37D). No differences were observed among other T cell subtypes in the different treatment groups. [Example]

[0418] N65S-1N-m3A7 / IL-15 (surrogate) efficacy study in multiple mouse syngeneic tumor models Mice were inoculated subcutaneously into the right flank with the indicated amount of tumor cells (Table 24) in 0.1 mL of PBS. Animals were randomized and tumors were grown until the mean tumor volume reached approximately 100 mm. 3 (75~125mm 3 Treatment was initiated when tumor volume reached a mean tumor size (between 100 and 150 mm). Tumor volumes were measured twice weekly using calipers in two dimensions, and volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (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 (tumor-free) were observed 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. A fusion protein comprising: Anti-PD-1 antibody; IL-15Rα sushi domain polypeptide; and comprising an IL-15 polypeptide, The fusion protein comprises a light chain sequence of SEQ ID NO:219, a first heavy chain sequence of SEQ ID NO:240 and a second heavy chain sequence of SEQ ID NO:

241.

2. The fusion protein of claim 1, wherein the fusion protein comprises no more than one IL-15Rα sushi domain polypeptide and no more than one IL-15 polypeptide.

3. The fusion protein of claim 1 , comprising two light chains and two heavy chains.

4. One or more expression vectors comprising a nucleic acid encoding the fusion protein according to any one of claims 1 to 3.

5. A cell comprising the expression vector described in claim 4.

6. (i) a fusion protein according to any one of claims 1 to 3; (ii) a pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising: