Anti-PD-1 Antibodies and Fusion Proteins

Abstract

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

1 - 76 . (canceled) 
     
     
         77 . An anti-PD-1 antibody, or an antigen-binding fragment thereof, wherein:
 a. the sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 21, 22, and 23; and   b. the sequences of CDR1L, CDR2L and CDR3L comprise:
 i. SEQ ID NOS: 96, 97 and 82 
 ii. SEQ ID NOS: 45, 46, and 47; 
 iii. SEQ ID NOS: 66, 67, and 68; 
 iv. SEQ ID NOS: 70, 67, and 71; 
 v. SEQ ID NOS: 73, 74, and 75; 
 vi. SEQ ID NOS: 77, 78, and 47; 
 vii. SEQ ID NOS: 80, 81, and 82; 
 viii. SEQ ID NOS: 77, 78, and 84; 
 ix. SEQ ID NOS: 77, 86, and 47; 
 x. SEQ ID NOS: 88, 89, and 47; 
 xi. SEQ ID NOS: 66, 67, and 47; 
 xii. SEQ ID NOS: 80, 92, and 75; 
 xiii. SEQ ID NOS: 80, 94, and 71; 
 xiv. SEQ ID NOS: 99, 100, and 47; 
 xv. SEQ ID NOS: 102, 103, and 104; 
 xvi. SEQ ID NOS: 106, 103, and 47; 
 xvii. SEQ ID NOS: 108, 103, and 47; 
 xviii. SEQ ID NOS: 110, 111, and 75; 
 xix. SEQ ID NOS: 77, 103, and 113; 
 xx. SEQ ID NOS: 77, 111, and 47; 
 xxi. SEQ ID NOS: 116, 67, and 47; 
 xxii. SEQ ID NOS: 118, 119, and 47; 
 xxiii. SEQ ID NOS: 80, 78, and 47; 
 xxiv. SEQ ID NOS: 122, 103 and 47; 
 xxv. SEQ ID NOS: 124, 125, and 75; 
 xxvi. SEQ ID NOS: 127, 38, and 68; 
 xxvii. SEQ ID NOS: 129, 130, and 47; or 
 xxviii. SEQ ID NOS: 132, 133, and 75. 
   
     
     
         78 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 , wherein:
 a. the heavy chain variable region comprises:
 i. SEQ ID NO: 24, or 
 ii. a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24; and 
   b. the light chain variable region comprises:
 i. SEQ ID NO: 48, 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, or 134, or 
 ii. a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NO: 48, 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, or 134. 
   
     
     
         79 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 , wherein:
 the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23   the sequence of CDR1L comprises the sequence of SEQ ID NO: 96;   the sequence of CDR2L comprises the sequence of SEQ ID NO: 97; and   the sequence of CDR3L comprises the sequence of SEQ ID NO: 82.   
     
     
         80 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 79 , wherein:
 a. the heavy chain variable region comprises SEQ ID NO: 24, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24; and   b. the light chain variable region comprises SEQ ID NO: 98, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 98.   
     
     
         81 . An anti-PD-1 antibody, or an antigen-binding fragment thereof, wherein:
 a. the sequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 17, 18, and 19; and   b. the sequences of CDR1L, CDR2L and CDR3L comprise:
 i. SEQ ID NOS: 41, 49, and 50; 
 ii. SEQ ID NOS: 41, 42, and 43; 
 iii. SEQ ID NOS: 41, 52, and 53; 
 iv. SEQ ID NOS: 41, 55, and 56; or 
 v. SEQ ID NOS: 58, 59, and 60. 
   
     
     
         82 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 81 , wherein:
 a. the heavy chain variable region comprises:
 i. SEQ ID NO: 20, or 
 ii. a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20; and 
   b. the light chain variable region comprises:
 i. SEQ ID NO: 44, 51, 54, 57, or 61, or 
 ii. a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 44, 51, 54, 57, or 61. 
   
     
     
         83 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 , wherein the antibody or antigen-binding fragment is a multispecific or a bispecific antibody or antigen-binding fragment thereof. 
     
     
         84 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 , wherein the antibody or antigen-binding fragment is an scFv, Fv, Fab′, Fab, F(ab′)2, or diabody. 
     
     
         85 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 , wherein the antibody is an IgG class immunoglobulin, optionally having isotype IgG1 or IgG4. 
     
     
         86 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 , wherein the antibody or antigen-binding fragment comprises a first and a second heavy chain constant region, and wherein the antibody or antigen-binding fragment thereof comprises at least one modification in the CH3 domains of the first and the second heavy chain causing heterodimerization. 
     
     
         87 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 86 , wherein:
 a. the CH3 domain of the first heavy chain comprises one or more of amino acid substitutions S354C and T366W; and the CH3 domain of the second heavy chain comprises one or more of amino acid substitutions Y349C, T366S, L368A, and Y407V;   b. the CH3 domain of the first heavy chain comprises one or more of amino acid substitutions T350V, L351Y, F405A, and Y407V; and the CH3 domain of the second heavy chain comprises one or more of amino acid substitutions T350V, T366L, K392L, and T394W; or   c. the CH3 domain of the first heavy chain comprises one or more of amino acid substitutions L351Y, F405A, and Y407V; and the CH3 domain of the second heavy chain comprises one or more of amino acid substitutions T366L, K392L, and T394W (Kabat EU index numbering).   
     
     
         88 . The anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 86 , wherein the Fc domain, if present, comprises one or more amino acid substitution that reduces binding to an Fc receptor or reduces effector function, wherein the one or more amino acid substitution is selected from L234A, L235A, and P329G (Kabat EU index numbering). 
     
     
         89 . A fusion protein comprising:
 a. the anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 ;   b. an IL-15Rα sushi domain polypeptide comprising the amino acid sequence of SEQ ID NO: 214, or an amino acid sequence that is at least 95% identical to SEQ ID NO:   214;   c. an IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 212, or an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 212; and   d. a first linker polypeptide joining the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide, optionally wherein the IL-15Rα sushi domain polypeptide is fused to the N-terminus of the IL-15 polypeptide.   
     
     
         90 . The fusion protein of  claim 89 , wherein (i) the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide are fused to (ii) the N-terminus of the heavy chain variable region, optionally by a second linker. 
     
     
         91 . The fusion protein of  claim 90 , wherein the first linker (i) is between 5 and 40 amino acids long, optionally, consists predominantly of Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L), and Gln (Q); and/or (ii) comprises a sequence selected from the group consisting of SEQ ID NOS: 215-218 and 244. 
     
     
         92 . The fusion protein of  claim 91 , wherein the second linker (i) is between 5 and 40 amino acids long, optionally, consists predominantly of Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L), and Gln (Q), and/or (ii) comprises a sequence selected from the group consisting of SEQ ID NOS: 215-218 and 244. 
     
     
         93 . The fusion protein of  claim 92 , wherein the fusion protein comprises not more than one IL-15Rα sushi domain polypeptide and not more than one IL-15 polypeptide. 
     
     
         94 . The fusion protein of  claim 92 , wherein the IL-15 polypeptide is a mutant IL-15 polypeptide comprising one or more amino acid substitutions at positions 45, 65, or 108 as numbered according to the mature human IL-15 sequence of SEQ ID NO: 212, wherein the one or more amino acid substitutions in the IL-15 polypeptide are selected from L45A, N65A, N65D, N65S, N65K, and Q108S as numbered according to the mature human IL-15 sequence of SEQ ID NO: 212. 
     
     
         95 . A fusion protein comprising a light chain sequence comprising SEQ ID NO: 219 or SEQ ID NO: 98 and a first heavy chain sequence comprising SEQ ID NO: 240 and a second heavy chain sequence comprising SEQ ID NO: 241. 
     
     
         96 . A nucleic acid sequence encoding the anti-PD-1 antibody, or an antigen-binding fragment thereof of  claim 77 . 
     
     
         97 . A cell comprising the nucleic acid of  claim 96 . 
     
     
         98 . A vector comprising nucleic acids encoding the light and heavy chains encoding the anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 . 
     
     
         99 . A pharmaceutical composition comprising (i) the anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 ; and (ii) a pharmaceutically acceptable carrier. 
     
     
         100 . A method of inhibiting binding of PD-1 to a ligand of PD-1, optionally PD-L1 or PD-L2, in a subject in need thereof, the method comprising administering to the subject an effective amount of the anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 . 
     
     
         101 . A method for increasing T cell activation or stimulating the immune system in a subject in need thereof, the method comprising administering to the subject an effective amount of the anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 . 
     
     
         102 . A method of treating cancer or reducing tumor growth or tumor metastasis in a subject in need thereof, the method comprising administering to the subject an effective amount of the anti-PD-1 antibody, or an antigen-binding fragment thereof, of  claim 77 . 
     
     
         103 . A method of inhibiting binding of PD-1 to a ligand of PD-1, optionally PD-L1 or PD-L2, in a subject in need thereof, the method comprising administering to the subject an effective amount of the fusion protein of  claim 89 . 
     
     
         104 . A method for increasing T cell activation or stimulating the immune system in a subject in need thereof, the method comprising administering to the subject an effective amount of the fusion protein of  claim 89 . 
     
     
         105 . A method of treating cancer or reducing tumor growth or tumor metastasis in a subject in need thereof, the method comprising administering to the subject an effective amount of fusion protein of  claim 89 .