Formulations of single domain antigen binding molecules

Abstract

The invention relates to formulations of single domain antigen binding molecules, e.g., nanobody molecules, in particular formulations of TNF-binding nanobody molecules. The single domain antigen binding molecules can include one or more single binding domains that interact with, e.g., bind to, one or more target proteins. The formulations are useful, e.g., as pharmaceutical formulations. Method of preparing, and using the formulations described herein, to treat, e.g., TNF-associated disorders, are also disclosed.

1 .- 30 . (canceled) 
     
     
         31 . A formulation comprising:
 (a) a single domain antigen binding (SDAB) molecule at a concentration from about 0.1 mg/mL to about 350 mg/mL, comprising one or more single domain molecules, wherein at least one of the single domain molecule binds to a serum protein;   (b) a lyoprotectant; and   (c) a histidine buffer at a concentration about 5 to about 50 mM, such that the pH of the formulation is about 5.0 to 7.5.   
     
     
         32 . The formulation of  claim 31 , wherein said serum protein is human serum albumin (HSA). 
     
     
         33 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA comprises three CDRs, wherein
 CDR1 has the amino acid sequence SFGMS (SEQ ID NO: 5), or an amino acid sequence that differs by 2 or 1 amino acid substitutions from SFGMS (SEQ ID NO:5);   CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 6), or an amino acid sequence that differs by 3, 2 or 1 amino acid substitutions from SISGSGSDTLYADSVKG (SEQ ID NO: 6); and   CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 7), or an amino acid sequence that differs by 2 or 1 amino acid substitutions from GGSLSR (SEQ ID NO: 7)   
     
     
         34 . The formulation of  claim 33 , wherein said amino acid substitutions are conservative substitutions. 
     
     
         35 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA has one or more biological activities of the HSA-binding single domain molecule shown in  FIG. 30  (SEQ ID NO: 1). 
     
     
         36 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA binds to the same epitope as the epitope recognized by the HSA-binding single domain molecule shown in  FIG. 30  (SEQ ID NO: 1). 
     
     
         37 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA binds to a similar epitope as the epitope recognized by the HSA-binding single domain molecule shown in  FIG. 30  (SEQ ID NO: 1). 
     
     
         38 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA has an activity similar to any of the HSA-binding single domain molecule disclosed in WO 06/122786. 
     
     
         39 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA has a binding affinity similar to any of the HSA-binding single domain molecule disclosed in WO 06/122786. 
     
     
         40 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA has a dissociation constant similar to any of the HSA-binding single domain molecule disclosed in WO 06/122786. 
     
     
         41 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA has a binding specificity similar to any of the HSA-binding single domain molecule disclosed in WO 06/122786. 
     
     
         42 . The formulation of  claim 31 , wherein the at least one single domain molecule that binds to a serum protein is selected from the group consisting of:
 single domain molecules comprising a variable region having the amino acid sequence from about amino acids 125 to 239 of  FIG. 30  (SEQ ID NO: 1),   single domain molecules comprising a variable region that differs by up to 10 amino acids from said variable region,   single domain molecules at least 85%, 90%, 95% or more identical relative to the amino acid sequence shown in  FIG. 30  (SEQ ID NO: 1), and   single domain molecules having up to 20, 15, 10, 5, 4, 3, 2, 1 amino acid changes relative to the amino acid sequence shown in  FIG. 30  (SEQ ID NO: 1).   
     
     
         43 . The formulation of  claim 31 , wherein the SDAB molecule is selected from the group consisting of monovalent, multivalent, bivalent, trivalent, tetravalent, monospecific, multispecific, bispecific, trispecific and tetraspecific SDAB molecules. 
     
     
         44 . The formulation of  claim 32 , wherein the at least one single domain molecule that binds to HSA is ALB1, ALB6, ALB7, ALB8, ALB9, or ALB10 disclosed in WO 06/122786. 
     
     
         45 . The formulation of  claim 31 , wherein said lyoprotectant is selected from the group consisting of sugars, optionally sucrose, sorbitol, and trehalose; amino acids, optionally monosodium glutamate and histidine; methylamines, optionally betaine; lyotrophic salts, optionally magnesium sulfate; polyols, optionally trihydric sugar alcohols and higher sugar alcohols, including glycerin, erythritol, glycerol, arabitol, xylitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof. 
     
     
         46 . The formulation of  claim 31 , wherein the SDAB molecule further comprises at least one, optionally two single domain molecules, that bind to tumor necrosis factor α (TNFα). 
     
     
         47 . The formulation of  claim 31 , wherein the SDAB molecule further comprises one or more single domain molecules that is useful in treating autoimmune disorders, optionally arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polyarticular juvenile idiopathic arthritis (JIA), psoriatic arthritis, lupus-associated arthritis, or ankylosing spondylitis; scleroderma; systemic lupus erythematosus; Sjogren's syndrome; vasculitis; multiple sclerosis; autoimmune thyroiditis dermatitis, optionally atopic dermatitis or eczematous dermatitis; myasthenia gravis; inflammatory bowel disease (IBD); Crohn's disease; (ulcerative) colitis; diabetes mellitus (type I); inflammatory conditions of the skin, optionally psoriasis; acute inflammatory conditions, optionally endotoxemia, sepsis, septicaemia, toxic shock syndrome, or infectious disease; transplant rejection; and allergy. 
     
     
         48 . A method or process of preparing a formulation of a SDAB molecule, comprising: expressing the SDAB molecule in a cell culture; purifying the SDAB molecule by passing the SDAB molecule through at least one of a chromatography purification step, or an ultrafiltration/diafiltration steps; adjusting the concentration of the SDAB molecule to about 0.1 to 350 mg/mL in a formulation containing a lyoprotectant at a concentration of about 5% to about 10%; and a Histidine buffer at a concentration about 5 to about 50 mM, such that the pH of the formulation is about 5 to 7.5. 
     
     
         49 . A method of preparing a reconstituted formulation containing a SDAB molecule, comprising: lyophilizing a mixture of a SDAB molecule and a lyoprotectant, and a buffer, thereby forming a lyophilized mixture; and reconstituting the lyophilized mixture in a diluent, thereby preparing the formulation, wherein the reconstituted formulation comprises (a) a SDAB molecule at a concentration of about 0.1 mg/mL to about 350 mg/mL; (b) a lyoprotectant at a concentration of about 5% to about 10%; (c) a Histidine buffer at a concentration about 5 to about 50 mM, such that the pH of the formulation is about 5.0 to 7.5. 
     
     
         50 . A kit or an article of manufacture, comprising a container containing the formulation of  claim 31 , and instructions for use. 
     
     
         51 . The kit or article of manufacture of  claim 50 , wherein the formulation is present in a vial or an injectable syringe. 
     
     
         52 . A method of treating or preventing a TNFα-related disorder, comprising administering to a subject, a pharmaceutical composition that comprises the formulation of  claim 46 , thereby reducing one or more symptoms associated with the TNFα-related disorder. 
     
     
         53 . The method of  claim 52 , wherein the TNFα-related disorder is an inflammatory or an autoimmune disorder. 
     
     
         54 . The method of  claim 53 , wherein the TNFα-related disorder is selected from rheumatoid arthritis (RA), arthritic conditions (e.g., psoriatic arthritis, polyarticular juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, or multiple sclerosis. 
     
     
         55 . A method of analyzing a manufacturing process, comprising: providing a sample of the formulation of  claim 31 ; assessing a parameter of the formulation selected from color, clarity, viscosity, or an amount of one or more HMW, LMW, acidic or basic species; determining whether the parameter meets a preselected criteria, thereby analyzing the process.