Covalently Bonded Diabodies Having Immunoreactivity with PD-1 and LAG-3, and Methods of Use Thereof

Abstract

The present invention is directed to bi-specific diabodies that comprise two or more polypeptide chains and which possess at least one Epitope-Binding Site that is immunospecific for an epitope of PD-1 and at least one Epitope-Binding Site that is immunospecific for an epitope of LAG-3 (i.e., a “PD-1×LAG-3 bi-specific diabody”). More preferably, the present invention is directed to bi-specific diabodies that comprise four polypeptide chains and which possess two Epitope-Binding Sites that are immunospecific for one (or two) epitope(s) of PD-1 and two Epitope-Binding Site that are immunospecific for one (or two) epitope(s) of LAG-3 (i.e., a “PD-1×LAG-3 bi-specific, tetra-valent diabody”). The present invention also is directed to such diabodies that additionally comprise an immunoglobulin Fc Domain (“bi-specific Fc diabodies and bi-specific, tetra-valent, Fc diabodies”). The diabodies of the present invention are capable of simultaneously binding to PD-1 and to LAG-3, particularly as such molecules are arrayed on the surfaces of human cells. The invention is directed to pharmaceutical compositions that contain such diabodies, and to methods involving the use of such diabodies in the treatment of cancer and other diseases and conditions.

1 . A method of treating cancer in a subject in need thereof, said method comprising administering to said subject an effective amount of a bi-specific Fc diabody capable of immunospecific binding to an epitope of PD-1 and to an epitope of LAG-3, wherein said diabody comprises first, second, third and fourth polypeptide chains, each having an amino terminus and a carboxy terminus, and wherein:
 (A) said first and second polypeptide chains are covalently bonded to one another, said first and third polypeptide chains are covalently bonded to one another, and said third and fourth polypeptide chains are covalently bonded to one another;   (B) said first and third polypeptide chains of said diabody each comprise, in the N-terminal to C-terminal direction, a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 or LAG-3, a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 or PD-1, a Heterodimer-Promoting Domain and a CH2-CH3 Domain, wherein said Light Chain Variable Domains and said Heavy Chain Variable Domains are incapable of associating to form an Epitope-Binding Site capable of binding an epitope of PD-1 or an epitope of LAG-3; and   (C) said second and said fourth polypeptide chains of said diabody each comprise, in the N-terminal to C-terminal direction, a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 or LAG-3, a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 or PD-1, and a Heterodimer-Promoting Domain, wherein said Light Chain Variable Domains and said Heavy Chain Variable Domains are incapable of associating to form an Epitope-Binding Site capable of binding an epitope of PD-1 or an epitope of LAG-3;   and wherein:   I. (1) said Light Chain Variable Domain of said first polypeptide chain and said Heavy Chain Variable Domain of said second polypeptide chain associate to form a first Epitope-Binding Site and said Heavy Chain Variable Domain of said first polypeptide chain and said Light Chain Variable Domain of said second polypeptide chain associate to form a second Epitope-Binding Site; and
 (2) said Light Chain Variable Domain of said third polypeptide chain and said Heavy Chain Variable Domain of said fourth polypeptide chain associate to form a third Epitope-Binding Site and said Heavy Chain Variable Domain of said third polypeptide chain and said Light Chain Variable Domain of said fourth polypeptide chain associate to form a fourth Epitope-Binding Site; 
 wherein:
 (i) said first and third formed Epitope-Binding Sites are capable of immunospecifically binding to an epitope of PD-1 and said second and fourth Epitope-Binding Sites are capable of immunospecifically binding to an epitope of LAG-3; or 
 (ii) said first and third Epitope-Binding Sites are capable of immunospecifically binding to an epitope of LAG-3 and said second and fourth Epitope-Binding Sites are capable of immunospecifically binding to an epitope of PD-1; and 
 
   II. (1) said Heterodimer-Promoting Domain of said first and third polypeptide chains each comprises an amino acid sequence of SEQ ID NO:16 and said Heterodimer-Promoting Domain of said second and fourth polypeptide chains each comprises an amino acid sequence of SEQ ID NO:17; or
 (2) said Heterodimer-Promoting Domain of said first and third polypeptide chains each comprises the amino acid sequence of SEQ ID NO:17 and said Heterodimer-Promoting Domain of said second and fourth polypeptide chains each comprises the amino acid sequence of SEQ ID NO:16; 
   III. said CH2-CH3 Domains of said first and third polypeptide chains associate to form an Fc Domain.   
     
     
         2 . The method of  claim 1 , wherein said CH2-CH3 Domains of said first and third polypeptide chains each comprises the amino acid sequence of SEQ ID NO:24. 
     
     
         3 . The method of  claim 1 , wherein:
 (a) said Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:11, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:12; and/or   (b) said Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1 comprises the amino acid sequence of SEQ ID NO:2, and said Light Chain Variable Domain of an antibody that is immunospecific for PD-1 comprises the amino acid sequence of SEQ ID NO:3.   
     
     
         4 . The method of  claim 2 , wherein:
 (a) said Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:11, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 comprises the amino acid sequence of SEQ ID NO:12; and/or   (b) said Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1 comprises the amino acid sequence of SEQ ID NO:2, and wherein said Light Chain Variable Domain of an antibody that is immunospecific for PD-1 comprises the amino acid sequence of SEQ ID NO:3.   
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein said cancer is an adrenal gland cancer, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's sarcoma, a kidney cancer, a leukemia, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a pheochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a renal metastatic cancer, a rhabdoid tumor, a rhabdomyosarcoma, a sarcoma, a skin cancer, a soft-tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a thyroid metastatic cancer, or a uterine cancer. 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein:
 (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3; and   (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1.   
     
     
         13 . The method of  claim 2 , wherein:
 (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3; and   (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1.   
     
     
         14 . The method diabody of  claim 1 , wherein:
 (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1; and   (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3.   
     
     
         15 . The method diabody of  claim 2 , wherein:
 (A) said first and third polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for LAG-3 and a Heavy Chain Variable Domain of an antibody that is immunospecific for PD-1; and   (B) said second and fourth polypeptide chains each comprise a Light Chain Variable Domain of an antibody that is immunospecific for PD-1 and a Heavy Chain Variable Domain of an antibody that is immunospecific for LAG-3.   
     
     
         16 . The method of  claim 1 , wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. 
     
     
         17 . The method of  claim 2 , wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. 
     
     
         18 . The method of  claim 12 , wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. 
     
     
         19 . The method of  claim 13 , wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. 
     
     
         20 . The method of  claim 14 , wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. 
     
     
         21 . The method of  claim 15 , wherein said Heavy Chain Variable Domain is separated from said Heterodimer-Promoting Domain in each of said first, second, third and fourth polypeptide chains by a linker comprising the amino acid sequence of SEQ ID NO:18. 
     
     
         22 . The method of  claim 1 , wherein said CH2-CH3 Domains of said first and third polypeptide chains are of the IgG1, IgG2, IgG3, or IgG4 isotype or a variant thereof. 
     
     
         23 . The method of  claim 22 , wherein the isotype is IgG4 or a variant thereof. 
     
     
         24 . The method of  claim 22 , wherein the isotype is IgG1 or a variant thereof. 
     
     
         25 . The method of  claim 24 , wherein said CH2-CH3 Domains of said first and third polypeptide chains each comprise the amino acid sequence of SEQ ID NO: 24.