Antibody Immune Cell Inhibitor Fusion Proteins

Abstract

The disclosure provides antibody immune cell inhibitor fusion proteins comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure also provides antibody immune cell inhibitor fusion proteins comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure further provides pharmaceutical compositions and kits that comprise such antibody immune cell inhibitor fusion proteins, and methods of treatment using such proteins.

1 . An antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site:
 wherein one polypeptide chain has a structure represented by the formula. II 1 -V L -C L -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -V H -C H1 -II 4 ;   wherein:   V L  is an immunoglobulin light chain variable domain;   V H  is an immunoglobulin heavy chain variable domain;   C L  is an immunoglobulin light chain constant domain;   C H1  is the immunoglobulin C H1  heavy chain constant domain;   II 1 , II 2 , II 3 , and II 4  are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;   wherein at least one of II 1 , II 2 , II 3 , and II 4  is an immune cell inhibitor domain of an immunoglobulin superfamily member; and   wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune cell effector cell only when bound to the target antigen at the antigen binding site.   
     
     
         2 . The antibody immune cell inhibitor fusion protein of  claim 1 , wherein the polypeptide chains comprise:
 (a) the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 17;   (b) the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 14;   (c) the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 14;   (d) the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 16;   (e) the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13;   (f) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4;   (g) the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4;   (h) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6;   (i) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5;   (j) the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10;   (k) the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;   (l) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11;   (m) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 12;   (n) the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28;   (o) the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 33;   (p) the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 34;   (q) the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 14;   (r) the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 14;   (s) the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 14;   (t) the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 14;   (u) the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 14;   (v) the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 14;   (w) the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 14;   (x) the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 14;   (y) the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 14; or   (z) the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 14.   
     
     
         3 . A pharmaceutical composition comprising a therapeutically effective amount of the antibody immune cell inhibitor fusion protein of  claim 2  and a pharmaceutically acceptable carrier. 
     
     
         4 . A kit comprising the antibody immune cell inhibitor fusion protein of  claim 2 . 
     
     
         5 . An antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site;
 wherein one polypeptide chain has a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -L 3 -V H -C H1 -L 4 -II 4 ;   wherein:   V L  is an immunoglobulin light chain variable domain;   V H  is an immunoglobulin heavy chain variable domain;   C L  is an immunoglobulin light chain constant domain;   C H1  is the immunoglobulin C H1  heavy chain constant domain; L 1 , L 2 , L 3 , and L 4  are each independently a linker domain or are absent; and   II 1 , II 2 , II 3 , and II 4  are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;   wherein at least one of L 1 , L 2 , L 3 , and L 4  is a linker domain and at least one of II 1 , II 2 , II 3 , and II 4  a is an immune cell inhibitor domain of an immunoglobulin superfamily member; and   wherein the antibody immune cell inhibiter fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.   
     
     
         6 . The antibody immune cell inhibitor fusion protein of  claim 1 , wherein the polypeptide chains comprise:
 (a) the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 17;   (b) the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 14;   (c) the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 14;   (d) the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 16;   (e) the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13;   (f) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4;   (g) the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4;   (h) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6;   (i) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5;   (j) the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10;   (k) the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;   (l) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11;   (m) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 12;   (n) the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28;   (o) the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 33;   (p) the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 34;   (q) the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 14;   (r) the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 14;   (s) the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 14;   (t) the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 14;   (u) the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 14;   (v) the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 14;   (w) the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 14;   (x) the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 14;   (y) the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 14; or   (z) the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 14.   
     
     
         7 . A pharmaceutical composition comprising a therapeutically effective amount of the antibody immune cell inhibitor fusion protein of  claim 6  and a pharmaceutically acceptable carrier. 
     
     
         8 . A kit comprising the antibody immune cell inhibitor fusion protein of  claim 6 . 
     
     
         9 . A method of treating a patient having primary biliary cholangitis, comprising:
 administering an antibody immune cell inhibitor fusion protein to the patient, wherein the antibody immune cell inhibitor fusion protein comprises:   (A) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -V L -C L , and two polypeptide chains have a structure represented by the formula II 3 -V H -C H1 -Fc; wherein: V L  is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; V H  is an immunoglobulin heavy chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; C L  is an immunoglobulin light chain constant domain; C H1  is the immunoglobulin C H1  heavy chain constant domain; Fc is the immunoglobulin hinge region and C H2  and C H3 immunoglobulin heavy chain constant domains; and II 1  and II 3  are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of II 1  and II 3  is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites;   (B) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -L 1 -V L -C L  and two polypeptide chains have a structure represented by the formula II 3 -L 3 -V H -C H1 -Fc; wherein: V L  is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; V H  is an immunoglobulin heavy chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; C L  is an immunoglobulin light chain constant domain; C H1  is the immunoglobulin C H1  heavy chain constant domain; Fc is the immunoglobulin hinge region and C H2  and C H3  immunoglobulin heavy chain constant domains; L 1  and L 3  are each independently a linker domain or are absent; and II 1  and II 3  are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of L 1  and L 3  is a linker domain and at least one of II 1  and II 3  is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites;   (C) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -V L -C L , and two polypeptide chains have a structure represented by the formula II 3 -V H -C H1 -Fc; wherein: V L  is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; V H  is an immunoglobulin heavy chain variable domain that specifically hinds to dihydrolipoamide acetyltransferase; C L  is an immunoglobulin light chain constant domain; C H1  is the immunoglobulin C H1  heavy chain constant domain; Fc is the immunoglobulin hinge region and C H2  and C H3  immunoglobulin heavy chain constant domains; and II 1  and II 3  are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of II 1  and II 3  is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites; or   (D) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -L 1 -V L -C L  and two polypeptide chains have a structure represented by the formula II 3 -L 3 -V H -C H1 -fc; wherein: V L  is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; is an immunoglobulin heavy chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; C L  is an immunoglobulin light chain constant domain; C H1  is the immunoglobulin C H1  heavy chain constant domain; Fc is the immunoglobulin hinge region and C H2  and C H3  immunoglobulin heavy chain constant domains; L 1  and L 3  are each independently a linker domain or are absent; and II 1  and II 3  are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of L 1  and L 3  is a linker domain and at least one of II 1  and II 3  is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites; and   wherein, for (A), (B), (C), and (D), the polypeptide chains comprise:   (a) the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 17;   (b) the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 14;   (c) the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 14;   (d) the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 16;   (e) the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13;   (f) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4;   (g) the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4;   (h) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6;   (i) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5;   (j) the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10;   (k) the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;   (l) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11;   (m) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 12;   (n) the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28;   (o) the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 33;   (p) the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 34;   (q) the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 14;   (r) the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 14;   (s) the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 14;   (t) the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 14;   (u) the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 14;   (v) the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 14;   (w) the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 14;   (x) the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 14;   (y) the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 14; or   (z) the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 14.   
     
     
         10 . The method of  claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein to the patient comprises administering a composition comprising the antibody immune cell inhibitor fusion protein. 
     
     
         11 . The method of  claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), and wherein each of II 1  and II 3  is an immune cell inhibitor domain. 
     
     
         12 . The method of  claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), and wherein at least one of II 1  and II 3  is an immune cell inhibitor domain and inhibits or diminishes activation of an immune effector cell involved in the immune response to self-tissue. 
     
     
         13 . The method of  claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), and wherein at least one of II 1  and II 3  is absent. 
     
     
         14 . The method of  claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), wherein the immune cell inhibitor domain comprises a Programmed Death Ligand 1 (PD-L1) domain, Programmed Death Ligand 2 (PD-L2) domain, B7 Homolog 3 (B7-H3) domain, B7 Homolog 4 (B7-H4) domain, Herpesvirus Entry Mediator (HVEM) domain, B7 Homolog 5 (B7-H5) domain, B7 Homolog 6 (B7-H6) domain, B7 Homolog 7 (B7-H7) domain, Cytotoxic T Lymphocyte-Associated 4 (CTLA-4) domain, CD200 domain, T-cell immunoglobulin or Mucin Domains-Containing Protein 3 (TIM-3) domain. 
     
     
         15 . The method of  claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (D), wherein at least one of L 1  and L 3  is absent. 
     
     
         16 . The method of  claim 9 , where the administering comprises parenteral administration. 
     
     
         17 . The method of  claim 9 , where the administering comprises the administration of a dosage from about 0.1 mg/kg to up to about 100 mg/kg of a composition comprising the antibody immune cell inhibitor fusion protein of (A), (B), (C), or (D). 
     
     
         18 . The method of  claim 17 , where the composition has a pH range from about 5 to about 8. 
     
     
         19 . The method of  claim 17 , where the administering comprises the administration of (A) or (B). 
     
     
         20 . The method of  claim 17 , where the administering comprises the administration of (C) or (D).