Dual Variable Domain Immunoglobulins and Uses Thereof

Abstract

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

We claim: 
     
         1 .- 36 . (canceled) 
     
     
         37 . An isolated nucleic acid encoding a binding protein amino acid sequence, wherein the binding protein comprises first and second polypeptide chains, each independently comprising VD1-(X1)n-VD2-C-(X2)n, wherein
 VD1 is a first variable domain;   VD2 is a second variable domain;   C is a constant domain;   X1 is a linker;   X2 is an Fc region;   n is 0 or 1;   
       wherein the VD1 domains on the first and second polypeptide chains form a first functional target binding site, and the VD2 domains on the first and second polypeptide chains forms a second functional target binding site, wherein the binding protein is capable of binding:
 (a) methotrexate (MTX) and CD-3, wherein
 (i) the variable domains that form a functional target binding site for MTX comprise
 three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and 
 
 (ii) the variable domains that form a functional target binding site for CD-3 comprise:
 three CDRs from SEQ ID NO: 32 and a three CDRs from SEQ ID NO: 33, 
 three CDRs from SEQ ID NO: 68 and three CDRs from SEQ ID NO: 69, or 
 three CDRs from SEQ ID NO: 100 and three CDRs from SEQ ID NO: 101; 
 
 and 
 (iii) the IC 50  of the binding pair is at most 4.0 nM as measured in a methotrexate neutralization assay; 
 
 (b) MTX and EGFR, wherein
 (i) the variable domains that form a functional target binding site for MTX comprise:
 three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and 
 
 (ii) the variable domains that form a functional target binding site for EGFR comprise:
 three CDRs from SEQ ID NO: 34 and three CDRs from SEQ ID NO: 35, 
 three CDRs from SEQ ID NO: 62 and three CDRs from SEQ ID NO: 63, or 
 three CDRs from SEQ ID NO: 76 and three CDRs from SEQ ID NO: 77; 
 
 and 
 (iii) the IC 50  of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay; 
 
 or 
 (c) MTX and HER-2, wherein
 (i) the variable domains that form a functional target binding site for MTX comprise:
 three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and 
 
 (ii) the variable domains that form a functional target binding site for HER-2 comprise:
 three CDRs from SEQ ID NO: 36 and three CDRs from SEQ ID NO: 37; or 
 three CDRs from SEQ ID NO: 98 and three CDRs from SEQ ID NO: 99; 
 
 and 
 (iii) the IC 50  of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay. 
 
 
     
     
         38 . A vector comprising an isolated nucleic acid according to  claim 37 . 
     
     
         39 . The vector according to  claim 38 , wherein said vector is selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO, and pBJ. 
     
     
         40 . A host cell comprising a vector according to  claim 38 , wherein the host cell is optionally selected from the group consisting of a prokaryotic cell,  Escherichia coli , a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a yeast cell, a  Saccharomyces cervisiae  cell, an Sf9 cell, a mammalian cell, an avian cell, an insect cell, a CHO cell, and a COS cell. 
     
     
         41 .- 50 . (canceled) 
     
     
         51 . A method of producing a binding protein, comprising culturing the host cell of  claim 40  under conditions sufficient to produce the binding protein. 
     
     
         52 .- 58 . (canceled) 
     
     
         59 . A method for treating a subject for a disease or a disorder by administering to the subject a binding protein such that treatment is achieved, wherein the binding protein comprises first and second polypeptide chains, each independently comprising VD1-(X1)n-VD2-C-(X2)n, wherein
 VD1 is a first variable domain;   VD2 is a second variable domain;   C is a constant domain;   X1 is a linker;   X2 is an Fc region;   n is 0 or 1;   
       wherein the VD1 domains on the first and second polypeptide chains form a first functional target binding site, and the VD2 domains on the first and second polypeptide chains form a second functional target binding site, wherein the binding protein is capable of binding:
 (a) methotrexate (MTX) and CD3, wherein
 (i) the variable domains that form a functional target binding site for MTX comprise
 three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and 
 
 (ii) the variable domains that form a functional target binding site for CD-3 comprise:
 three CDRs from SEQ ID NO: 32 and a three CDRs from SEQ ID NO: 33, 
 three CDRs from SEQ ID NO: 68 and three CDRs from SEQ ID NO: 69, or 
 three CDRs from SEQ ID NO: 100 and three CDRs from SEQ ID NO: 101; 
 
 and 
 (iii) the IC 50  of the binding pair is at most 4.0 nM as measured in a methotrexate neutralization assay; 
 
 (b) MTX and EGFR, wherein
 (i) the variable domains that form a functional target binding site for MTX comprise:
 three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and 
 
 (ii) the variable domains that form a functional target binding site for EGFR comprise:
 three CDRs from SEQ ID NO: 34 and three CDRs from SEQ ID NO: 35, 
 three CDRs from SEQ ID NO: 62 and three CDRs from SEQ ID NO: 61, or 
 three CDRs from SEQ ID NO: 76 and three CDRs from SEQ ID NO: 77; 
 
 and 
 (ii) the IC 50  of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay; 
 
 or 
 (c) MTX and HER-2, wherein
 (i) the variable domains that form a functional target binding site for MTX comprise:
 three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and 
 
 (ii) the variable domains that form a functional target binding site for HER-2 comprise:
 three CDRs from SEQ ID NO: 36 and three CDRs from SEQ ID NO: 37; or 
 three CDRs from SEQ ID NO: 98 and three CDRs from SEQ ID NO: 99; 
 
 and 
 (iii) the IC 50  of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay. 
 
 
     
     
         60 . The method of  claim 59 , wherein said disorder is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and  salmonella  associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-I autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, cholestatis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma) Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection,  legionella , leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphedema, malaria, malignant Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache, mitochondrial multi.system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,  mycobacterium avium intracellulare, mycobacterium tuberculosis , myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral ateriosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia,  pneumocystis carinii  pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive Supranuclear Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis/aseptic meningitis, viral-associated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still's disease, alopecia areata, anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia, arteriosclerosis, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with  streptococcus  infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (CIS) with risk for multiple sclerosis, conjunctivitis, childhood onset psychiatric disorder, chronic obstructive pulmonary disease (COPD), dacryocystitis, dermatomyositis, diabetic retinopathy, diabetes mellitus, disk herniation, disk prolaps, drug induced immune hemolytic anemia, endocarditis, endometriosis, endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major, gestational pemphigoid, Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis, keratoconjunctivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, morbus bechterev, motor neuron disorders, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery, disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome, primary Parkinsonism, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), prostatitis, pure red cell aplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis), scleroderma, secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal insufficiency, silicone associated connective tissue disease, sneddon-wilkinson dermatosis, spondilitis ankylosans, Stevens-Johnson syndrome (SJS), systemic inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor, type 1 allergic reaction, type II diabetes, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, wound healing, yersinia and  salmonella  associated arthropathy. 
     
     
         61 . The method according to  claim 60 , wherein said administering to the subject is by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. 
     
     
         62 .- 79 . (canceled) 
     
     
         80 . A binding protein comprising first and second polypeptide chains, each independently comprising-VD1-(X1)n-VD2-C-(X2)n, wherein
 VD1 is a first variable domain;   VD2 is a second variable domain;   C is a constant domain;   X1 is a linker; and   X2 is an Fc region;   
       wherein the VD1 domains on the first and second polypeptide chains form a first functional target binding site and the VD2 domains on the first and second polypeptide chains form a second functional target binding site, and wherein the binding protein is capable of binding
 (a) methotrexate (MTX) and mCD-3, wherein the binding protein comprises:
 DVD1011 (comprising SEQ ID NOs: 110 and 111), 
 DVD1012 (comprising SEQ ID NOs: 112 and 113), 
 DVD1013 (comprising SEQ ID NOs: 114 and 115), 
 DVD1014 (comprising SEQ ID NOs: 116 and 117), 
 DVD1015 (comprising SEQ ID NOs: 118 and 119), 
 DVD1016 (comprising SEQ ID NOs: 120 and 121), 
 DVD1017 (comprising SEQ ID NOs: 122 and 123), or 
 DVD1018 (comprising SEQ ID NOs: 124 and 125); 
 
 (b) methotrexate (MTX) and mCD-19, wherein the binding protein comprises:
 DVD1019 (comprising SEQ ID NOs: 126 and 127), 
 DVD1020 (comprising SEQ ID NOs: 128 and 129), 
 DVD1021 (comprising SEQ ID NOs: 130 and 131), 
 DVD1022 (comprising SEQ ID NOs: 132 and 133), 
 DVD1023 (comprising SEQ ID NOs: 134 and 135), 
 DVD1024 (comprising SEQ ID NOs: 136 and 137), 
 DVD1025 (comprising SEQ ID NOs: 138 and 139), or 
 DVD1026 (comprising SEQ ID NOs: 140 and 141); 
 
 (c) methotrexate (MTX) and hCD-3, wherein the binding protein comprises:
 DVD1027 (comprising SEQ ID NOs: 142 and 143), 
 DVD1028 (comprising SEQ ID NOs: 144 and 145), 
 DVD1029 (comprising SEQ ID NOs: 146 and 147), 
 DVD1030 (comprising SEQ ID NOs: 148 and 149), 
 DVD1031 (comprising SEQ ID NOs: 150 and 151), 
 DVD1032 (comprising SEQ ID NOs: 152 and 153), 
 DVD1033 (comprising SEQ ID NOs: 154 and 155), 
 DVD1034 (comprising SEQ ID NOs: 156 and 157), 
 DVD1216 (comprising SEQ ID NOs: 186 and 187), or 
 DVD1217 (comprising SEQ ID NOs: 188 and 189), 
 
 (d) methotrexate (MTX) and hCD-19, wherein the binding protein comprises:
 DVD1035 (comprising SEQ ID NOs: 158 and 159), 
 DVD1036 (comprising SEQ ID NOs: 160 and 161), 
 DVD1037 (comprising SEQ ID NOs: 162 and 163), 
 DVD1038 (comprising SEQ ID NOs: 164 and 165), 
 DVD1039 (comprising SEQ ID NOs: 166 and 167), 
 DVD1040 (comprising SEQ ID NOs: 168 and 169), 
 DVD1041 (comprising SEQ ID NOs: 170 and 171), or 
 DVD1042 (comprising SEQ ID NOs: 172 and 173); 
 
 (e) methotrexate (MTX) and hCD-20, wherein the binding protein comprises:
 DVD1208 (comprising SEQ ID NOs: 174 and 175), or 
 DVD1209 (comprising SEQ ID NOs: 176 and 177); 
 
 (f) methotrexate (MTX) and EGFR, wherein the binding protein comprises:
 DVD1210 (comprising SEQ ID NOs: 178 and 179), or 
 DVD1211 (comprising SEQ ID NOs: 180 and 181); 
 
 or 
 (g) methotrexate (MTX) and HER-2, wherein the binding protein comprises
 DVD1212 (comprising SEQ ID NOs: 182 and 183), or 
 DVD1213 (comprising SEQ ID NOs: 184 and 185).