VARIABLE SINGLE-DOMA ANTIBODIES AGAINST OX40L, CONSTRUCTS AND THEIR THERAPEUTIC USE

Description

FIELD OF THE INVENTION

[0001] The present invention is defined in the appended claims and relates to an immunoglobulin single variable domain that can specifically bind to OX40L, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such immunoglobulin single variable domains (also referred to herein as "amino acid sequences of the invention", "compounds of the invention", and "polypeptides of the invention", respectively).

[0002] The invention also relates to nucleic acids encoding such immunoglobulin single variable domains and polypeptides (also referred to herein as "nucleic acids of the invention" or "nucleotide sequences of the invention"); to methods for preparing such immunoglobulin single variable domains and polypeptides; to host cells expressing or capable of expressing such immunoglobulin single variable domains or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such immunoglobulin single variable domains, polypeptides, nucleic acids and / or host cells; and to uses of such immunoglobulin single variable domains or polypeptides, nucleic acids, host cells and / or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.BACKGROUND OF THE INVENTION

[0003] Allergic diseases have reached epidemic proportions worldwide and are generally accepted to be increasing. Allergy symptoms are treated efficiently with cheap small molecule drugs in most patients, but none are curative. However, there is a clear unmet medical need for patients suffering persistent asthma. Current standard treatment for inflammatory diseases include immunosuppressing or immunomodulating medications such as (but not limited to) corticosteroids, non-steroidal anti inflammatory drugs, TNF-alpha antagonists, cytokine(receptor) agonists or antagonists. Current standard treatment for allergic asthma include inhaled or systemically administered corticosteroids in combination with inhaled (long or short acting) Beta-2 agonists, possibly combined with medications such as (but not limited to) Leukotriene receptor antagonists, theophylline, cromolyn, nedocromyl.

[0004] The introduction of monoclonal antibodies and soluble cytokine receptors is revolutionizing approaches to the treatment of asthma and allergy. The successful introduction of omalizumab (anti IgE) for severe allergic asthma has stimulated great interest in this approach, but even with this humanised monoclonal antibody, cost effectiveness analyses are restricting its use even though it has passed scrutiny by such agents as the National Institute of Health & Clinical Excellence in the UK. Also, substantial portions of the potential patient population in asthma have IgE levels higher than can successfully be neutralized with the current molecule. A more potent inhibitor is required for these, and attempts have been made to produce such a molecule.

[0005] Understanding the underlying mechanisms of allergic disease has stimulated the further development of a series of biologics targeted towards critical cells and molecules. Because of the sentinel roles of Th2 cells and their products, Th2 cytokines, in orchestrating allergic inflammation, they and their receptors are key therapeutic targets. One of these is the TNF family member "OX40L" (Review: Michael J. Gough et al., Therapeutic Targets of the TNF superfamily, edited by Iqbal S. Grewal, 2007). OX40L and antibodies against OX40L which are able to disrupt binding of OX40L to its receptor OX40 (and thus inhibit the relevant signaling cascade) are mentioned in e.g. WO95 / 12673, WO95 / 21915, WO99 / 15200, WO2006 / 029879, WO2005 / 094879 and WO2007 / 133290.

[0006] WO 2006 / 030220 A1 describes antibody polypeptides that monovalently bind to CD40L.

[0007] WO 2006 / 029879 A2 describes mAb LC001 which is also used as comparator in the experimental section of the present application. The invention is defined by the appended claims. Any other references to aspects or embodiments of the invention that do not fall within their scope are for illustrative purposes only.

[0008] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy (or for diagnosis).SUMMARY OF THE INVENTION

[0009] Single variable domains, such as Nanobodies and dAbs and antigen binding fragments derived therefrom are widely used to specifically target their respective antigens in research and therapeutic applications. However, Nanobodies and dAbs lack the Fc part and will not induce effector function mediated depletion of OX40L-expressing cells and thus potential toxicity associated with effector function mediated depletion is not of relevance. However, in order to compensate for the efficacy increase by the effector function of the full length monoclonal antibody (lacking in the single variable domains such as Nanobodies), there is then a need for an ultrapotent OX40L binding Nanobody or construct thereof. Furthermore, the patient group with severe persistent allergic asthma who remain symptomatic despite optimized standard treatment, require significant use of health services and are at risk of severe exacerbations including morbidity. Thus, new therapies to address these clear and unmet needs need to be developed.

[0010] The invention has overcome the disadvantages of the prior art and addresses the unmet medical needs.

[0011] In particular, the invention relates to an immunoglobulin single variable domain that can specifically bind to OX40L represented by SEQ ID NO: 175, in which: CDR1 is the amino acid sequence of SEQ ID NO: 135; and CDR2 is the amino acid sequence of SEQ ID NO: 149; and CDR3 is the amino acid sequence of SEQ ID NO: 163, wherein said immunoglobulin single variable domain has at least 80% amino acid identity to SEQ ID NO: 181, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the following amino acid residues: at position 11: L, S, V, M, W, F, T, Q E, A, R, G, K, Y, N, P, I, preferably L, at position 37: F, Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F or Y, at position 44: E, Q, G, D, A, K, R, L, P, S, V, H, T, N, W, M, I, preferably G, E or Q, most preferably G at position 45: L, R, P, H, F, G, Q, S, E, T, Y, C, I, D, V preferably L or R, at position 47: F, L or W, G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D, preferably W, L or F, at position 83: R, K, T, E, Q, N, S, I, V, G, M, L, A, D, Y, H, preferably K or R; most preferably K at position 84: P, S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E, preferably P at position 103: W, R, G, S, K, A, M, Y, L, F, T, N, V, Q, P, E, C, preferably W at position 104: G, A, S, T, D, P, N, E, C, L, preferably G at position 108: Q, L, R, P, E, K, S, T, M, A, H, preferably Q or L.

[0012] The invention also relates to a polypeptide that comprises or essentially consists of one or more immunoglobulin single variable domains as defined in the appended claims. Moreover, the invention relates to a nucleic acid or nucleotide sequence that encodes an immunoglobulin single variable domain or a polypeptide as defined in the appended claims as well as to a composition comprising at least one immunoglobulin single variable domain, polypeptide or nucleic acid or nucleotide sequence as defined in the appended claims. In addition, the invention also provides a method for producing an immunoglobulin single variable domain or a polypeptide as defined in the appended claims. Furthermore, the immunoglobulin single variable domain, polypeptide or composition is for use as a medicament, optionally, for the use in prevention and / or treatment of at least one inflammatory disease and / or disorder, asthma, allergic asthma, chronic colitis, Crohn's disease, inflammatory bowel disease, and / or arthrosclerosis as defined in the appended claims.

[0013] The following description is considered useful for understanding the invention.

[0014] The polypeptides and compositions of the present description can generally be used to modulate, and in particular inhibit and / or prevent, binding of OX40L to OX40, and thus to modulate, and in particular inhibit or prevent, the signalling that is mediated by OX40L and / or OX40, to modulate the biological pathways in which OX40L and / or OX40 are involved, and / or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways.

[0015] As such, the polypeptides and compositions of the present description can be used for the prevention and treatment (as defined herein) of inflammatory disorders such as e.g. asthma and / or allergic asthma. Generally, "inflammatory disorders such as e.g. asthma" can be defined as diseases and disorders that can be prevented and / or treated, respectively, by suitably administering to a subject in need thereof (i.e. having the disease or disorder or at least one symptom thereof and / or at risk of attracting or developing the disease or disorder) of either a polypeptide or composition of the description (and in particular, of a pharmaceutically active amount thereof) and / or of a known active principle active against OX40L or a biological pathway or mechanism in which OX40L is involved (and in particular, of a pharmaceutically active amount thereof). Examples of such inflammatory disorders such as e.g. asthma and / or allergic asthma, will be clear to the skilled person based on the disclosure herein, and for example include the following diseases and disorders: rheumatoid arthritis, asthma, allergic asthma including moderate to severe asthma such as e.g. severe persistent asthma whose symptoms are not adequately controlled with inhaled corticosteroids.

[0016] In particular, the polypeptides and compositions of the present description can be used for the prevention and treatment of inflammatory disorders such as e.g. asthma and / or allergic asthma which are characterized by excessive and / or unwanted signaling mediated by OX40L or by the pathway(s) in which OX40L is involved. Examples of such inflammatory disorders such as e.g. asthma and / or allergic asthma (Gough, supra), chronic colitis such as Crohn's disease (Tostuka et al., 2003, Am J Physiol Gastrointest. Liver Physiol. 284, G595-G603), inflammatory bowel disease (Souza H S et al., 1999, Gut, 45, 856-863), and / or atherosclerosis (Olofsson P.S., 2008, Circulation, 117, 1291-1301) will again be clear to the skilled person. Without being limiting, other diseases and / or disorders associated with OX40L includean immune disorder and / or an autoimmune disorder (such as asthma, allergic asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, graft verses host disease (GVHD), and / or systemic lupus erythematosus); a disorder associated with virus, bacteria or other infectious agent, for example virus- induced imunopathology, e.g., pathology induced by infection with influenze or RSV or related viruses, e.g., in the lung; arthritis (acute and chronic, rheumatoid arthritis including juvenile-onset rheumatoid arthritis and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen- induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis / rheumatoid spondylitis); autoimmune lymphoproliferative disease, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, hives, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, gastrointestinal inflammation, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, graft-versus-host disease, angioedema such as hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN (RPGN), proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, food allergies, drug allergies, insect allergies, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, auto-immune asthma, allergic asthma, and pediatric asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-O blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric IDDM, adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic large-artery disorder, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large- vessel vasculitis (including polymyalgia rheumatica and giant-cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex- mediated diseases, anti-glomerular basement membrane disease, anti- phospholipid antibody syndrome, motoneuritis, allergic neuritis, Behcet's disease / syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury due to an autoimmune condition, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, neuroinflammatory disorders, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia including, for example, idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird- fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis (systemic inflammatory response syndrome (SIRS)), endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity pneumonitis, conjunctivitis, such as vernal catarrh, keratoconjunctivitis sicca, and epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular insufficiency) such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte- adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndromes, including polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, allergic sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, spondyloarthropathies, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis. Other examples, which in some cases encompass those listed above, include but are not limited to autoimmune rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis / dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteriitis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant. GVHD, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).

[0017] Thus, without being limited thereto, the amino acid sequences and polypeptides of the description can for example be used to prevent and / or to treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate OX40L-mediated signalling, such as those mentioned in the prior art cited above. It is also envisaged that the polypeptides of the description can be used to prevent and / or to treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or will be proposed or developed in future. In addition, it is envisaged that, because of their favourable properties as further described herein, the polypeptides of the present description may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and / or that the polypeptides of the present description may provide new methods and regimens for treating the diseases and disorders described herein.

[0018] Other applications and uses of the amino acid sequences and polypeptides of the description will become clear to the skilled person from the further disclosure herein.

[0019] Generally, it is an object of the description to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the diagnosis, prevention and / or treatment of inflammatory disorders such as e.g. asthma and / or allergic asthma and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and / or treatment of such diseases and disorders that involve the administration and / or use of such agents and compositions.

[0020] In particular, it is an object of the description to provide such pharmacologically active agents, compositions and / or methods that have certain advantages compared to the agents, compositions and / or methods that are currently used and / or known in the art. These advantages will become clear from the further description below.

[0021] More in particular, it is an object of the description to provide therapeutic proteins that can be used as pharmacologically active agents, as well as compositions comprising the same, for the diagnosis, prevention and / or treatment of inflammatory disorders such as e.g. asthma and / or allergic asthma and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and / or treatment of such diseases and disorders that involve the administration and / or the use of such therapeutic proteins and compositions.

[0022] Accordingly, it is a specific object of the present description to provide amino acid sequences that are directed against (as defined herein) OX40L, in particular against OX40L from a warm-blooded animal, more in particular against OX40L from a mammal such as e.g. cynomolgus OX40L (SEQ ID NO: 176), and especially against human OX40L (SEQ ID NO: 175); and to provide proteins and polypeptides comprising or essentially consisting of at least one such amino acid sequence.

[0023] In particular, it is a specific object of the present description to provide such amino acid sequences and such proteins and / or polypeptides that are suitable for prophylactic, therapeutic and / or diagnostic use in a warm-blooded animal, and in particular in a mammal, and more in particular in a human being.

[0024] More in particular, it is a specific object of the present description to provide such amino acid sequences and such proteins and / or polypeptides that can be used for the prevention, treatment, alleviation and / or diagnosis of one or more diseases, disorders or conditions associated with OX40L and / or mediated by OX40L (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

[0025] It is also a specific object of the description to provide such amino acid sequences and such proteins and / or polypeptides that can be used in the preparation of pharmaceutical or veterinary compositions for the prevention and / or treatment of one or more diseases, disorders or conditions associated with and / or mediated by OX40L (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

[0026] In the description, generally, these objects are achieved by the use of the amino acid sequences, proteins, polypeptides and compositions that are described herein.

[0027] In general, the description provides amino acid sequences that are directed against (as defined herein) and / or can specifically bind (as defined herein) to OX40L; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

[0028] More in particular, the description provides amino acid sequences that can bind to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

[0029] In particular, amino acid sequences and polypeptides of the description are preferably such that they: bind to OX40L with a dissociation constant (K D ) of 10 -5< to 10 -12< moles / liter or less, and preferably 10 -7< to 10 -12< moles / liter or less and more preferably 10 -8< to 10 -12< moles / liter (i.e. with an association constant (K A ) of 10 5< to 10 12< liter / moles or more, and preferably 10 7< to 10 12< liter / moles or more and more preferably 10 8< to 10 12< liter / moles); and / or such that they: bind to OX40L with a k on -rate of between 10 2< M -1< s -1< to about 10 7< M -1< s -1< , preferably between 10 3< M -1< s -1< and 10 7< M -1< s -1< , more preferably between 10 4< M -1< s -1< and 10 7< M -1< s -1< , such as between 10 5< M -1< s -1< and 10 7< M -1< s -1< ; and / or such that they: bind to OX40L with a k off rate between 1 s -1< (t 1 / 2 =0.69 s) and 10 -6< s -1< (providing a near irreversible complex with a t 1 / 2 of multiple days), preferably between 10 -2< s -1< and 10 -6< s -1< , more preferably between 10 -3< s -1< and 10 -6< s -1< , such as between 10 -4< s -1< and 10 -6< s -1< .

[0030] Preferably, a monovalent amino acid sequence of the description (or a polypeptide that contains only one amino acid sequence of the description) is preferably such that it will bind to OX40L with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

[0031] Some preferred IC50 values for binding of the amino acid sequences or polypeptides of the description to OX40L will become clear from the further description and examples herein.

[0032] For binding to OX40L, an amino acid sequence of the description will usually contain within its amino acid sequence one or more amino acid residues or one or more stretches of amino acid residues (i.e. with each "stretch" comprising two or amino acid residues that are adjacent to each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the description can bind to OX40L, which amino acid residues or stretches of amino acid residues thus form the "site" for binding to OX40L (also referred to herein as the "antigen binding site").

[0033] The amino acid sequences provided by the description are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the description (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the description and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the description may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than OX40L), so as to provide a monovalent, multivalent or multispecific polypeptide of the description, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).

[0034] The amino acid sequences and polypeptides of the description as such preferably essentially consist of a single amino acid chain that is not linked via disulphide bridges to any other amino acid sequence or chain (but that may or may not contain one or more intramolecular disulphide bridges. For example, it is known that Nanobodies - as described herein - may sometimes contain a disulphide bridge between CDR3 and CDR1 or FR2). However, it should be noted that one or more amino acid sequences of the description may be linked to each other and / or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the description (for example Fab' fragments, F(ab') 2 fragments, ScFv constructs, "diabodies" and other multispecific constructs. Reference is for example made to the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep; 23(9):1126-36).

[0035] Generally, when an amino acid sequence of the description (or a compound, construct or polypeptide comprising the same) is intended for administration to a subject (for example for therapeutic and / or diagnostic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject; or, when it does occur naturally in said subject, in essentially isolated form (as defined herein).

[0036] It will also be clear to the skilled person that for pharmaceutical use, the amino acid sequences of the description (as well as compounds, constructs and polypeptides comprising the same) are preferably directed against human OX40L; whereas for veterinary purposes, the amino acid sequences and polypeptides of the description are preferably directed against OX40L from the species to be treated, or at least cross-reactive with OX40L from the species to be treated.

[0037] Furthermore, an amino acid sequence of the description may optionally, and in addition to the at least one binding site for binding against OX40L, contain one or more further binding sites for binding against other antigens, proteins or targets.

[0038] The efficacy of the amino acid sequences and polypeptides of the description, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include Ascaris suum model for asthma in monkey, as well as the assays and animal models used in the experimental part below and in the prior art cited herein.

[0039] Also, according to the description, amino acid sequences and polypeptides that are directed against OX40L from a first species of warm-blooded animal may or may not show cross-reactivity with OX40L from one or more other species of warm-blooded animal. For example, amino acid sequences and polypeptides directed against human OX40L may or may not show cross reactivity with OX40L from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and / or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) and / or with OX40L from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with OX40L (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug development point of view, since it allows the amino acid sequences and polypeptides against human OX40L to be tested in such disease models.

[0040] More generally, amino acid sequences and polypeptides of the description that are cross-reactive with OX40L from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species. Thus, it is also encompassed within the scope of the description that amino acid sequences and polypeptides directed against OX40L from one species of animal (such as amino acid sequences and polypeptides against human OX40L) can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and / or polypeptides provide the desired effects in the species to be treated.

[0041] The present description is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of OX40L against which the amino acid sequences and polypeptides of the description are directed. For example, the amino acid sequences and polypeptides may or may not be directed against an "interaction site" (as defined herein). However, it is generally assumed and preferred that the amino acid sequences and polypeptides of the description are preferably directed against an interaction site (as defined herein), and in particular against one or more epitopes of Nanobodies as described herein, e.g. one or more epitopes of Nanobody 01B11 (SEQ ID NO: 179), 18E09 (SEQ ID NO: 183) and 15B07 (SEQ ID NO: 182). Thus, in one preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the description are directed against one or more epitopes of Nanobody 01B11 (SEQ ID NO: 179), 18E09 (SEQ ID NO: 183) and 15B07 (SEQ ID NO: 182) and are as further defined herein.

[0042] As further described herein, a polypeptide of the description may contain two or more amino acid sequences of the description that are directed against OX40L. Generally, such polypeptides will bind to OX40L with increased avidity compared to a single amino acid sequence of the description. Such a polypeptide may for example comprise two amino acid sequences of the description that are directed against the same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of OX40L (which may or may not be an interaction site); or comprise at least one "first" amino acid sequence of the description that is directed against a first antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of OX40L (which may or may not be an interaction site); and at least one "second" amino acid sequence of the description that is directed against a second antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) different from the first (and which again may or may not be an interaction site). Preferably, in such "biparatopic" polypeptides of the description, at least one amino acid sequence of the description is directed against an interaction site (as defined hereindescription.

[0043] Also, when the target is part of a binding pair (for example, a receptor-ligand binding pair), the amino acid sequences and polypeptides may be such that they compete with the cognate binding partner (e.g. the ligand, receptor or other binding partner, as applicable) for binding to the target, and / or such that they (fully or partially) neutralize binding of the binding partner to the target.

[0044] It is also within the scope of the description that, where applicable, an amino acid sequence of the description can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confirmations of OX40L. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of OX40L to which the amino acid sequences and / or polypeptides of the description bind may be essentially the same (for example, if OX40L contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the description may bind to such different antigenic determinants, epitopes, parts, domains, subunits of OX40L with an affinity and / or specificity which may be the same or different). Also, for example, when OX40L exists in an activated conformation and in an inactive conformation, the amino acid sequences and polypeptides of the description may bind to either one of these confirmation, or may bind to both these confirmations (i.e. with an affinity and / or specificity which may be the same or different). Also, for example, the amino acid sequences and polypeptides of the description may bind to a conformation of OX40L in which it is bound to a pertinent ligand, may bind to a conformation of OX40L in which it not bound to a pertinent ligand, or may bind to both such conformations (again with an affinity and / or specificity which may be the same or different).

[0045] It is also expected that the amino acid sequences and polypeptides of the description will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of OX40L; or at least to those analogs, variants, mutants, alleles, parts and fragments of OX40L that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the description bind in OX40L (e.g. in wild-type OX40L). Again, in such a case, the amino acid sequences and polypeptides of the description may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and / or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the description bind to (wild-type) OX40L. It is also included within the scope of the description that the amino acid sequences and polypeptides of the description bind to some analogs, variants, mutants, alleles, parts and fragments of OX40L, but not to others.

[0046] When OX40L exists in a monomeric form and in one or more multimeric forms, it is within the scope of the description that the amino acid sequences and polypeptides of the description only bind to OX40L in monomeric form, only bind to OX40L in multimeric form, or bind to both the monomeric and the multimeric form. Again, in such a case, the amino acid sequences and polypeptides of the description may bind to the monomeric form with an affinity and / or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the description bind to the multimeric form.

[0047] Also, when OX40L can associate with other proteins or polypeptides to form protein complexes (e.g. with multiple subunits), it is within the scope of the description that the amino acid sequences and polypeptides of the description bind to OX40L in its non-associated state, bind to OX40L in its associated state, or bind to both. In all these cases, the amino acid sequences and polypeptides of the description may bind to such multimers or associated protein complexes with an affinity and / or specificity that may be the same as or different from (i.e. higher than or lower than) the affinity and / or specificity with which the amino acid sequences and polypeptides of the description bind to OX40L in its monomeric and non-associated state.

[0048] Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against OX40L may bind with higher avidity to OX40L than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of OX40L may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against OX40L may (and usually will) bind also with higher avidity to a multimer of OX40L.

[0049] Generally, amino acid sequences and polypeptides of the description will at least bind to those forms of OX40L (including monomeric, multimeric and associated forms) that are the most relevant from a biological and / or therapeutic point of view, as will be clear to the skilled person.

[0050] It is also within the scope of the description to use parts, fragments, analogs, mutants, variants, alleles and / or derivatives of the amino acid sequences and polypeptides of the description, and / or to use proteins or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogs, mutants, variants, alleles and / or derivatives, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles and / or derivatives will usually contain (at least part of) a functional antigen-binding site for binding against OX40L; and more preferably will be capable of specific binding to OX40L, and even more preferably capable of binding to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and / or polypeptides will become clear from the further description herein. Additional fragments or polypeptides of the description may also be provided by suitably combining (i.e. by linking or genetic fusion) one or more (smaller) parts or fragments as described herein.

[0051] In one specific, but non-limiting aspect of the description, which will be further described herein, such analogs, mutants, variants, alleles, derivatives have an increased half-life in serum (as further described herein) compared to the amino acid sequence from which they have been derived. For example, an amino acid sequence of the description may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the description with increased half-life.

[0052] In one specific, but non-limiting aspect, the amino acid sequence of the description may be an amino acid sequence that comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold (i.e. by folding). Reference is inter alia made to the review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably, when properly folded so as to form an immunoglobulin fold, such an amino acid sequence is capable of specific binding (as defined herein) to OX40L; and more preferably capable of binding to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein. Also, parts, fragments, analogs, mutants, variants, alleles and / or derivatives of such amino acid sequences are preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.

[0053] In particular, but without limitation, the amino acid sequences of the description may be amino acid sequences that essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein).

[0054] The amino acid sequences of the description may in particular be an immunoglobulin sequence or a suitable fragment thereof, and more in particular be an immunoglobulin variable domain sequence, immunoglobulin single variable domain sequences or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a V H -sequence) or a suitable fragment thereof. When the amino acid sequence of the description is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a V H sequence that is derived from a human antibody) or be a so-called V HH -sequence (as defined herein) that is derived from a so-called "heavy chain antibody" (as defined herein).

[0055] However, it should be noted that the description is not limited as to the origin of the amino acid sequence of the description (or of the nucleotide sequence of the description used to express it), nor as to the way that the amino acid sequence or nucleotide sequence of the description is (or has been) generated or obtained. Thus, the amino acid sequences of the description may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences. In a specific but non-limiting aspect of the description, the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V HH sequences or Nanobodies), "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein.

[0056] Similarly, the nucleotide sequences of the description may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

[0057] The amino acid sequence of the description may in particular be an immunoglobulin single variable domain sequence such as a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a V HH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as to for example WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present description because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05 / 18629).

[0058] In particular, the amino acid sequence of the description may be a Nanobody ®< (as defined herein) or a suitable fragment thereof. [Note: Nanobody ®< , Nanobodies ®< and Nanoclone ®< are registered trademarks of Ablynx N.V.] Such Nanobodies directed against OX40L will also be referred to herein as "Nanobodies of the description".

[0059] For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described Nanobodies of the so-called "V H 3 class" (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V H 3 class such as DP-47, DP-51 or DP-29), which Nanobodies form a preferred aspect of this description. It should however be noted that the description in its broadest sense generally covers any type of Nanobody directed against OX40L, and for example also covers the Nanobodies belonging to the so-called "V H 4 class" (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V H 4 class such as DP-78), as for example described in WO 07 / 118670.

[0060] Generally, Nanobodies (in particular V HH sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more of the framework sequences (again as further described herein).

[0061] Thus, generally, a Nanobody can be defined as an amino acid sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

[0062] In particular, a Nanobody can be an amino acid sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

[0063] More in particular, a Nanobody can be an amino acid sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 below; and in which: ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded.

[0064] In these Nanobodies, the CDR sequences are generally as further defined herein.

[0065] Thus, the description also relates to such Nanobodies that can bind to (as defined herein) and / or are directed against OX40L, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such Nanobodies and / or suitable fragments.

[0066] SEQ ID NO's: 179 to 185 (see Table A-1) give the amino acid sequences of a number of V HH sequences that have been raised against OX40L.

[0067] In particular, the description in some specific aspects provides: amino acid sequences that are directed against (as defined herein) OX40L and that have at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1). These amino acid sequences may further be such that they neutralize binding of the cognate ligand or receptor to OX40L; and / or compete with the cognate ligand or receptor for binding to OX40L; and / or are directed against an interaction site (as defined herein) on OX40L (such as the ligand or receptor binding site); amino acid sequences that cross-block (as defined herein) the binding of at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1) to OX40L and / or that compete with at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1) for binding to OX40L. Again, these amino acid sequences may further be such that they neutralize binding of the cognate ligand or receptor to OX40L; and / or compete with the cognate ligand or receptor for binding to OX40L; and / or are directed against an interaction site (as defined herein) on OX40L (such as the ligand or receptor binding site); which amino acid sequences may be as further described herein (and may for example be Nanobodies); as well as polypeptides of the description that comprise one or more of such amino acid sequences (which may be as further described herein, and may for example be bispecific and / or biparatopic polypeptides as described herein), and nucleic acid sequences that encode such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.

[0068] In some other specific aspects, the description provides: amino acid sequences of the description that are specific for OX40L compared to other members of the TNF family; which amino acid sequences of the description may be as further described herein (and may for example be Nanobodies); as well as polypeptides of the description that comprise one or more of such amino acid sequences (which may be as further described herein, and may for example be bispecific and / or biparatopic polypeptides as described herein), and nucleic acid sequences that encode such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.

[0069] Accordingly, some particularly preferred Nanobodies of the description are Nanobodies which can bind (as further defined herein) to and / or are directed against to OX40L and which: i) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1), in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded. In this respect, reference is also made to Table B-1, which lists the framework 1 sequences (SEQ ID NO's: 126 to 132), framework 2 sequences (SEQ ID NO's: 140 to 146), framework 3 sequences (SEQ ID NO's: 154 to 160) and framework 4 sequences (SEQ ID NO's: 168 to 174) of the Nanobodies of SEQ ID NO's: 179 to 185 (see Table A-1) (with respect to the amino acid residues at positions 1 to 4 and 27 to 30 of the framework 1 sequences, reference is also made to the comments made below. Thus, for determining the degree of amino acid identity, these residues are preferably disregarded); and in which: ii) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 below.

[0070] In these Nanobodies, the CDR sequences are generally as further defined herein.

[0071] Again, such Nanobodies may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V HH sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized" (as defined herein) Nanobodies, "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a Nanobody comprises a V HH sequence, said Nanobody may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized Nanobodies of the description. Similarly, when a Nanobody comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), said Nanobody may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized Nanobodies of the description.

[0072] In particular, humanized Nanobodies may be amino acid sequences that are as generally defined for Nanobodies in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and / or that corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V HH sequence with the corresponding framework sequence of one or more closely related human V H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V HH sequence (in any manner known per se, as further described herein) and the resulting humanized V HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and / or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody may be partially humanized or fully humanized.

[0073] Some particularly preferred humanized Nanobodies of the description are humanized variants of the Nanobodies of SEQ ID NO's: 179 to 185 (see Table A-1), of which the amino acid sequences of SEQ ID NO's: 199 to 226 (see Table A-2) are some especially preferred examples.

[0074] According to another specific aspect of the description, the description provides a number of stretches of amino acid residues (i.e. small peptides) that are particularly suited for binding to OX40L. These stretches of amino acid residues may be present in, and / or may be incorporated into, an amino acid sequence of the description, in particular in such a way that they form (part of) the antigen binding site of an amino acid sequence of the description. As these stretches of amino acid residues were first generated as CDR sequences of heavy chain antibodies or V HH sequences that were raised against OX40L (or may be based on and / or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as "CDR sequences" (i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). It should however be noted that the description in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in an amino acid sequence of the description, as long as these stretches of amino acid residues allow the amino acid sequence of the description to bind to OX40L. Thus, generally, the description in its broadest sense comprises any amino acid sequence that is capable of binding to OX40L and that comprises one or more CDR sequences as described herein, and in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire amino acid sequence forms a binding domain and / or binding unit that is capable of binding to OX40L. It should however also be noted that the presence of only one such CDR sequence in an amino acid sequence of the description may by itself already be sufficient to provide an amino acid sequence of the description that is capable of binding to OX40L; reference is for example again made to the so-called "Expedite fragments" described in WO 03 / 050531 or WO2009 / 127691.

[0075] Thus, in another specific, but non-limiting aspect, the amino acid sequence of the description may be an amino acid sequence that comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). In particular, an amino acid sequence of the description may be an amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof).

[0076] Generally, in this aspect of the description, the amino acid sequence of the description may be any amino acid sequence that comprises at least one stretch of amino acid residues, in which said stretch of amino acid residues has an amino acid sequence that corresponds to the sequence of at least one of the CDR sequences described herein. Such an amino acid sequence may or may not comprise an immunoglobulin fold. For example, and without limitation, such an amino acid sequence may be a suitable fragment of an immunoglobulin sequence that comprises at least one such CDR sequence, but that is not large enough to form a (complete) immunoglobulin fold (reference is for example again made to the "Expedite fragments" described in WO03 / 050531 or WO2009 / 127691). Alternatively, such an amino acid sequence may be a suitable "protein scaffold" that comprises at least one stretch of amino acid residues that corresponds to such a CDR sequence (i.e. as part of its antigen binding site). Suitable scaffolds for presenting amino acid sequences will be clear to the skilled person, and for example comprise, without limitation, to binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affibodies ™< ), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

[0077] Again, any amino acid sequence of the description that comprises one or more of these CDR sequences is preferably such that it can specifically bind (as defined herein) to OX40L, and more in particular such that it can bind to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein), that is as defined herein.

[0078] More in particular, the amino acid sequences according to this aspect of the description may be any amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least two amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that (i) when the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein or the CDR3 sequences described herein; (ii) when the first amino acid sequence is chosen from the CDR2 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein; or (iii) when the first amino acid sequence is chosen from the CDR3 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein.

[0079] Even more in particular, the amino acid sequences of the description may be amino acid sequences that comprise at least one antigen binding site, wherein said antigen binding site comprises at least three amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein, and the third amino acid sequence is chosen from the CDR3 sequences described herein. Preferred combinations of CDR1, CDR2 and CDR3 sequences will become clear from the further description herein. As will be clear to the skilled person, such an amino acid sequence is preferably an immunoglobulin sequence (as further described herein), but it may for example also be any other amino acid sequence that comprises a suitable scaffold for presenting said CDR sequences.

[0080] Thus, in one specific, but non-limiting aspect, the description relates to an amino acid sequence directed against OX40L, that comprises one or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; or any suitable combination thereof.

[0081] When an amino acid sequence of the description contains one or more amino acid sequences according to b) and / or c): i) any amino acid substitution in such an amino acid sequence according to b) and / or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein); and / or ii) the amino acid sequence according to b) and / or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a); and / or iii) the amino acid sequence according to b) and / or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0082] Similarly, when an amino acid sequence of the description contains one or more amino acid sequences according to e) and / or f): i) any amino acid substitution in such an amino acid sequence according to e) and / or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein); and / or ii) the amino acid sequence according to e) and / or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d); and / or iii) the amino acid sequence according to e) and / or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0083] Also, similarly, when an amino acid sequence of the description contains one or more amino acid sequences according to h) and / or i): i) any amino acid substitution in such an amino acid sequence according to h) and / or i) is preferably, and compared to the corresponding amino acid sequence according to g), a conservative amino acid substitution, (as defined herein); and / or ii) the amino acid sequence according to h) and / or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g); and / or iii) the amino acid sequence according to h) and / or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0084] It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the description that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

[0085] In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of: i) the amino acid sequences of SEQ ID NO's: 133 to 139; ii) the amino acid sequences of SEQ ID NO's: 147 to 153; and iii) the amino acid sequences of SEQ ID NO's: 161 to 167; or any suitable combination thereof.

[0086] Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against OX40L.

[0087] In a more specific, but again non-limiting aspect, the description relates to an amino acid sequence directed against OX40L, that comprises two or more stretches of amino acid residues chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).

[0088] In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of: i) the amino acid sequences of SEQ ID NO's: 133 to 139; ii) the amino acid sequences of SEQ ID NO's: 147 to 153; and iii) the amino acid sequences of SEQ ID NO's: 161 to 167; such that, (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 133 to 139, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 147 to 153 or of SEQ ID NO's: 161 to 167; (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 147 to 153, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 133 to 139 or of SEQ ID NO's: 161 to 167; or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 161 to 167, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 133 to 139 or of SEQ ID NO's: 147 to 153.

[0089] Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against OX40L.

[0090] In an even more specific, but non-limiting aspect, the description relates to an amino acid sequence directed against OX40L, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; the second stretch of amino acid residues is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; and the third stretch of amino acid residues is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167.

[0091] Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 133 to 139; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 147 to 153; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 161 to 167.

[0092] Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against OX40L.

[0093] Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

[0094] Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 179 to 185 (see Table A-1), in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the description can be as further described herein.

[0095] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to OX40L; and more in particular bind to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein.

[0096] When the amino acid sequence of the description essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the description is preferably such that: CDR1 is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; and / or CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; and / or CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167.

[0097] In particular, such an amino acid sequence of the description may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 133 to 139; and / or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 147 to 153; and / or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 161 to 167.

[0098] In particular, when the amino acid sequence of the description essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the description is preferably such that: CDR1 is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; and CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; and CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; or any suitable fragment of such an amino acid sequence

[0099] In particular, such an amino acid sequence of the description may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 133 to 139; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 147 to 153; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 161 to 167.

[0100] Again, preferred combinations of CDR sequences will become clear from the further description herein.

[0101] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to OX40L; and more in particular bind to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein.

[0102] In one preferred, but non-limiting aspect, the description relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 179 to 185 (see Table A-1), in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the description can be as further described herein.

[0103] In such an amino acid sequence of the description, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

[0104] The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V L -sequence) and / or from a heavy chain variable domain (e.g. a V H -sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V HH -sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional V H sequences that have been camelized (as defined herein).

[0105] The framework sequences are preferably such that the amino acid sequence of the description is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); is a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody); is a "dAb" (or an amino acid sequence that is suitable for use as a dAb); or is a Nanobody (including but not limited to V HH sequence). Again, suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

[0106] In particular, the framework sequences present in the amino acid sequences of the description may contain one or more of Hallmark residues (as defined herein), such that the amino acid sequence of the description is a Nanobody. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0107] Again, as generally described herein for the amino acid sequences of the description, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and / or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived). Such fragments may also again be such that they comprise or can form an immunoglobulin fold, or alternatively be such that they do not comprise or cannot form an immunoglobulin fold.

[0108] In one specific aspect, such a fragment comprises a single CDR sequence as described herein (and in particular a CDR3 sequence), that is flanked on each side by (part of) a framework sequence (and in particular, part of the framework sequence(s) that, in the immunoglobulin sequence from which the fragment is derived, are adjacent to said CDR sequence. For example, a CDR3 sequence may be preceded by (part of) a FR3 sequence and followed by (part of) a FR4 sequence). Such a fragment may also contain a disulphide bridge, and in particular a disulphide bridge that links the two framework regions that precede and follow the CDR sequence, respectively (for the purpose of forming such a disulphide bridge, cysteine residues that naturally occur in said framework regions may be used, or alternatively cysteine residues may be synthetically added to or introduced into said framework regions). For a further description of these "Expedite fragments", reference is again made to WO 03 / 050531, as well as to the US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" of Ablynx N.V. (inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; and Hoogenboom, Hendricus Renerus Jacobus Mattheus) filed on December 5, 2006 (see alsoWO2008 / 068280).

[0109] In another aspect, the description relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a "compound of the description" or "polypeptide of the description", respectively) that comprises or essentially consists of one or more amino acid sequences of the description (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. As will become clear to the skilled person from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the description (and / or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the description.

[0110] For example, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"'s, amino acid sequences that are suitable for use as a dAb, or Nanobodies.

[0111] Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and / or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the description so as to provide a "derivative" of an amino acid sequence or polypeptide of the description, as further described herein.

[0112] Also within the scope of the present description are compounds or constructs, that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences.

[0113] In the compounds or constructs described above, the one or more amino acid sequences of the description and the one or more groups, residues, moieties or binding units may be linked directly to each other and / or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide).

[0114] As will be clear from the further description above and herein, this means that the amino acid sequences of the description can be used as "building blocks" to form polypeptides of the description, i.e. by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as, without limitations, the biparatopic. bi / multivalent and bi / multispecific polypeptides of the description described herein) which combine within one molecule one or more desired properties or biological functions.

[0115] The compounds or polypeptides of the description can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the description to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the description. Polypeptides of the description can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the description, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the description. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.

[0116] The process of designing / selecting and / or preparing a compound or polypeptide of the description, starting from an amino acid sequence of the description, is also referred to herein as "formatting" said amino acid sequence of the description; and an amino acid of the description that is made part of a compound or polypeptide of the description is said to be "formatted" or to be "in the format of" said compound or polypeptide of the description. Examples of ways in which an amino acid sequence of the description can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the description.

[0117] In one specific aspect of the description, a compound of the description or a polypeptide of the description may have an increased half-life, compared to the corresponding amino acid sequence of the description. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the description that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the description that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the description that comprise at least one amino acid sequence of the description that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the description. Examples of polypeptides of the description that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the description are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb"'s, amino acid sequences that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to the further description and references mentioned herein); polypeptides in which an amino acid sequence of the description is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the description are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91 / 01743, WO 01 / 45746, WO 02 / 076489 and to the US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" of Ablynx N.V. filed on December 5, 2006 (see also WO2008 / 068280).

[0118] Generally, the compounds or polypeptides of the description with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the description per se. For example, the compounds or polypeptides of the description with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the description per se.

[0119] In a preferred, but non-limiting aspect of the description, such compounds or polypeptides of the description have a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the description per se.

[0120] In another preferred, but non-limiting aspect of the description, such compounds or polypeptides of the description exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the description may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

[0121] In another aspect, the description relates to a nucleic acid that encodes an amino acid sequence of the description or a polypeptide of the description (or a suitable fragment thereof). Such a nucleic acid will also be referred to herein as a "nucleic acid of the description" and may for example be in the form of a genetic construct, as further described herein.

[0122] In another aspect, the description relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the description and / or a polypeptide of the description; and / or that contains a nucleic acid of the description. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

[0123] The description further relates to a product or composition containing or comprising at least one amino acid sequence of the description, at least one polypeptide of the description (or a suitable fragment thereof) and / or at least one nucleic acid of the description, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

[0124] The description also relates to the use of an amino acid sequence, Nanobody or polypeptide of the description, or of a composition comprising the same, in (methods or compositions for) modulating OX40L, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from an inflammatory disorder such as e.g. asthma and / or allergic asthma).

[0125] The description also relates to methods for modulating OX40L, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from an inflammatory disorder such as e.g. asthma and / or allergic asthma), which method comprises at least the step of contacting OX40L with at least one amino acid sequence, Nanobody or polypeptide of the description, or with a composition comprising the same, in a manner and in an amount suitable to modulate OX40L with at least one amino acid sequence, Nanobody or polypeptide of the description.

[0126] The description also relates to the use of an amino acid sequence, Nanobody or polypeptide of the description in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating OX40L, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from an inflammatory disorder such as e.g. asthma and / or allergic asthma).

[0127] In the context of the present description, "modulating" or "to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of OX40L, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, "modulating" or "to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of OX40L, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of OX40L in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the description.

[0128] As will be clear to the skilled person, "modulating" may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and / or selectivity of OX40L for one or more of its targets, ligands or substrates; and / or effecting a change (which may either be an increase or a decrease) in the sensitivity of OX40L for one or more conditions in the medium or surroundings in which OX40L is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the description. As will be clear to the skilled person, this may again be determined in any suitable manner and / or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.

[0129] "Modulating" may also mean effecting a change (i.e. an activity as an agonist or as an antagonist, respectively) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which OX40L (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and / or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody or polypeptide of the description.

[0130] Modulating may for example involve reducing or inhibiting the binding of OX40L to one of its substrates or ligands and / or competing with a natural ligand, substrate for binding to OX40L. Modulating may also involve activating OX40L or the mechanism or pathway in which it is involved. Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.

[0131] The description further relates to methods for preparing or generating the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

[0132] Generally, these methods may comprise the steps of: a) providing a set, collection or library of amino acid sequences; and b) screening said set, collection or library of amino acid sequences for amino acid sequences that can bind to and / or have affinity for OX40L; and c) isolating the amino acid sequence(s) that can bind to and / or have affinity for OX40L.

[0133] In such a method, the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naïve set, collection or library of immunoglobulin sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and / or a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

[0134] Also, in such a method, the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as V H domains or V HH domains) or of light chain variable domains. For example, the set, collection or library of amino acid sequences may be a set, collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

[0135] In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with OX40L or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

[0136] In the above methods, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0137] In another aspect, the method for generating amino acid sequences comprises at least the steps of: a) providing a collection or sample of cells expressing amino acid sequences; b) screening said collection or sample of cells for cells that express an amino acid sequence that can bind to and / or has affinity for OX40L; and c) either (i) isolating said amino acid sequence; or (ii) isolating from said cell a nucleic acid sequence that encodes said amino acid sequence, followed by expressing said amino acid sequence.

[0138] For example, when the desired amino acid sequence is an immunoglobulin sequence, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a mammal that has been suitably immunized with OX40L or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

[0139] The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05 / 19824, WO 04 / 051268 and WO 04 / 106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).

[0140] In another aspect, the method for generating an amino acid sequence directed against OX40L may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and / or has affinity for OX40L; and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.

[0141] In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and / or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

[0142] Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V H domains or V HH domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

[0143] In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with OX40L or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

[0144] The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V HH sequences.

[0145] In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0146] In another aspect, the method for generating an amino acid sequence directed against OX40L may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and / or has affinity for OX40L and that is cross-blocked or is cross blocking a Nanobody of the description, e.g. one of SEQ ID NO's: 179 to 185 (Table A-1), or a humanized or sequence optimized Nanobody of the description such as e.g. one of SEQ ID NO's: 199 to 226 (Table A-2), or a polypeptide or construct of the description, e.g. one of SEQ ID NO's: 186 to 198 and 227 to 234 (see Table A-3 and Table A-4); and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.

[0147] The description also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

[0148] Also, following the steps above, one or more amino acid sequences of the description may be suitably humanized (or alternatively camelized); and / or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the description. Also, a nucleic acid sequence encoding an amino acid sequence of the description may be suitably humanized (or alternatively camelized) and suitably expressed; and / or one or more nucleic acid sequences encoding an amino acid sequence of the description may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the description.

[0149] The description further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and / or treatment for diseases and disorders associated with OX40L. Some preferred but non-limiting applications and uses will become clear from the further description herein.

[0150] The description also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy.

[0151] In particular, the description also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of a disease or disorder that can be prevented or treated by administering, to a subject in need thereof, of (a pharmaceutically effective amount of) an amino acid sequence, compound, construct or polypeptide as described herein.

[0152] More in particular, the description relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of inflammatory disorders such as e.g. asthma and / or allergic asthma.

[0153] Other aspects, embodiments, advantages and applications of the description will also become clear from the further description herein, in which the description will be described and discussed in more detail with reference to the Nanobodies of the description and polypeptides of the description comprising the same, which form some of the preferred aspects of the description.

[0154] As will become clear from the further description herein, Nanobodies generally offer certain advantages (outlined herein) compared to "dAb's" or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the Nanobodies of the description. However, it will be clear to the skilled person that the more general aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the description.DESCRIPTION OF THE FIGURES

[0155] Figure 1: Specificity of OX40L blocking Nanobody OX40L01E07. Binding of the OX40L01E07 to hOX40L captured on neutravidin was only inhibited by exogenously added hOX40L (o, dashed line) and was not affected by the addition of the other TNF superfamily members, i.e. hRANKL (■), hCD30L (▲ ), hCD40L (▼), hTRAIL (¨) or hTNFa (·). Figure 2: Inhibition of OX40L mediated effector memory T-cell activation by monovalent and trivalent bispecific anti-OX40L Nanobodies. 2x10 5< CD45RA- CCR7- effector memory T-cells, 1x10 4< hOX40L expressing CHO cells and dilution series of anti-OX40L Nanobodies or anti-OX40L benchmark were incubated for 3 days at 37°C. IL-4 production was measured in ELISA. All monovalent Nanobodies and benchmark Fab are shown with dotted lines and open symbols: OX40L01E07 (□), OX40L18E09 (∘) and OX40L19D08 ( -< ) in Fig2A; OX40L01E10 (□), OX40L15B07 (∘), OX40L01B11 ( -< ) in Fig2B, benchmark Fab (r). All trivalent bispecific Nanobodies and the benchmark IgG are shown with solid lines and symbols: OX40L003 (■), OX40L004 (·) and OX40L005 (◆) in Fig2A; OX40L006 (■), OX40L007 (·) and OX40L009 (¨) in Fig. 2B, benchmark IgG (▲). DETAILED DESCRIPTION OF THE DESCRIPTION

[0156] In the present description, examples and claims: a) Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks mentioned in paragraph a) on page 46 of WO 08 / 020079. b) Unless indicated otherwise, the terms "immunoglobulin sequence", "sequence", "nucleotide sequence" and "nucleic acid" are as described in paragraph b) on page 46 of WO 08 / 020079. c) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins. d) Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is made for example to Table A-2 on page 48 of the International application WO 08 / 020079 of Ablynx N.V. entitled "Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with Il-6 mediated signalling". e) For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated or determined as described in paragraph e) on page 49 of WO 08 / 020079, such as by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference at a single nucleotide (position); or using a suitable computer algorithm or technique, again as described in paragraph e) on pages 49 of WO 08 / 020079. f) For the purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence (also referred to herein as "amino acid identity") may be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08 / 020079, such as by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. as an "amino acid difference" as defined herein; or using a suitable computer algorithm or technique, again as described in paragraph f) on pages 49 and 50 of WO 08 / 020079.

[0157] Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, as described on page 50 of WO 08 / 020079.

[0158] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986. Information on the primary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V HH domain from a llama is for example given by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional V H domains form the V H / V L interface and potential camelizing substitutions on these positions can be found in the prior art cited above. g) Amino acid sequences and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length. h) When comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences. i) When a nucleotide sequence or amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of" another nucleotide sequence or amino acid sequence, this has the meaning given in paragraph i) on pages 51-52 of WO 08 / 020079. j) The term "in essentially isolated form" has the meaning given to it in paragraph j) on pages 52 and 53 of WO 08 / 020079. k) The terms "domain" and "binding domain" have the meanings given to it in paragraph k) on page 53 of WO 08 / 020079. I) The term "variable domain" refers to the part or domain of an immunoglobulin molecule or antibody which is partially or fully responsible for antigen binding. m) The term "single variable domain" or "immunoglobulin single variable domain", defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two "variable domains" interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.

[0159] In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. The term "immunoglobulin single variable domain" does comprise fragments of conventional immunoglobulins wherein the antigen binding site is formed by a single variable domain.

[0160] Generally, immunoglobulin single variable domains will be amino acid sequences that essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's). Such immunoglobulin single variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold. As such, the immunoglobulin single variable domain may for example comprise a light chain variable domain sequence (e.g. a V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a V H -sequence or V HH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit that essentially consists of the immunoglobulin single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and scFv fragments that need to interact with another variable domain - e.g. through a V H / V L interaction - to form a functional antigen binding domain).

[0161] In one aspect of the description, the immunoglobulin single variable domains are light chain variable domain sequences (e.g. a V L -sequence), or heavy chain variable domain sequences (e.g. a V H -sequence); more specifically, the single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.

[0162] The immunoglobulin single variable domain may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody ®< (as defined herein, and including but not limited to a V HH sequence) [Note: Nanobody ®< and Nanobodies ®< are registered trademarks of Ablynx N.V.]; other immunoglobulin single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited herein, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. 1989 (Nature 341: 544-546), to Holt et al. 2003 (Trends Biotechnol. 21: 484-490); as well as to for example WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present description because they are not of mammalian origin, immunoglobulin single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05 / 18629). n) The terms "antigenic determinant" and "epitope", which may also be used interchangeably herein, have the meanings given to it in paragraph l) on page 53 of WO 08 / 020079. o) As further described in paragraph m) on page 53 of WO 08 / 020079, an amino acid sequence (such as a Nanobody, an antibody, a polypeptide of the description, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and / or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against" or "directed against" said antigenic determinant, epitope, antigen or protein. p) The term "specificity" has the meaning given to it in paragraph n) on pages 53-56 of WO 08 / 020079; and as mentioned therein refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a Nanobody or a polypeptide of the description) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity, as described on pages 53-56 of WO 08 / 020079, which also describes some preferred techniques for measuring binding between an antigen-binding molecule (such as a Nanobody or polypeptide of the description) and the pertinent antigen. Typically, antigen-binding proteins (such as the amino acid sequences, Nanobodies and / or polypeptides of the description) will bind to their antigen with a dissociation constant (K D ) of 10 -5< to 10 -12< moles / liter or less, and preferably 10 -7< to 10 -12< moles / liter or less and more preferably 10 -8< to 10 -12< moles / liter (i.e. with an association constant (K A ) of 10 5< to 10 12< liter / moles or more, and preferably 10 7< to 10 12< liter / moles or more and more preferably 10 8< to 10 12< liter / moles). Any K D value greater than 10 4< mol / liter (or any K A value lower than 10 4< M -1< ) liters / mol is generally considered to indicate non-specific binding. Preferably, a monovalent immunoglobulin sequence of the description will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein. As will be clear to the skilled person, and as described on pages 53-56 of WO 08 / 020079, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned on pages 53-56 of WO 08 / 020079. q) The half-life of an amino acid sequence, compound or polypeptide of the description can generally be defined as described in paragraph o) on page 57 of WO 08 / 020079 and as mentioned therein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the description can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally be as described in paragraph o) on page 57 of WO 08 / 020079. As also mentioned in paragraph o) on page 57 of WO 08 / 020079, the half-life can be expressed using parameters such as the t1 / 2-alpha, t1 / 2-beta and the area under the curve (AUC). Reference is for example made to the Experimental Part below, as well as to the standard handbooks, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The terms "increase in half-life" or "increased half-life" as also as defined in paragraph o) on page 57 of WO 08 / 020079 and in particular refer to an increase in the t1 / 2-beta, either with or without an increase in the t1 / 2-alpha and / or the AUC or both. r) In the context of the present description, "modulating" or "to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay. In particular, "modulating" or "to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the description.

[0163] As will be clear to the skilled person, "modulating" may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and / or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and / or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the description. As will be clear to the skilled person, this may again be determined in any suitable manner and / or using any suitable assay known per se, depending on the target or antigen involved.

[0164] "Modulating" may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and / or using any suitable (in vitro and usually cellular or in assay) assay known per se, depending on the target or antigen involved. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the description.

[0165] Modulating may for example also involve allosteric modulation of the target or antigen; and / or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and / or competing with a natural ligand, substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions).

[0166] Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner. s) In respect of a target or antigen, the term "interaction site" on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an "interaction site" can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the description can bind such that the target or antigen (and / or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein). t) An amino acid sequence or polypeptide is said to be "specific for" a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a K D value, K A value, K off rate and / or K on rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10.000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide. For example, the first antigen may bind to the target or antigen with a K D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10.000 times less or even less than that, than the K D with which said amino acid sequence or polypeptide binds to the second target or polypeptide. Preferably, when an amino acid sequence or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen. u) The terms "cross-block", "cross-blocked" and "cross-blocking" are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a Nanobody, polypeptide or compound or construct of the description) to interfere with the binding of other amino acid sequences or binding agents of the description to a given target. The extend to which an amino acid sequence or other binding agents of the description is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block according to the description, can be determined using competition binding assays. One particularly suitable quantitative cross-blocking assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequences or other binding agents in terms of their binding to the target.

[0167] The following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the description. It will be appreciated that the assay can be used with any of the amino acid sequences or other binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross-blocking assay, the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target. Typically 200- 800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross-block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence. The concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip. The amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each amino acid sequence when passed over the target surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two amino acid sequences are cross-blocking each other. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the description is one which will bind to the target in the above Biacore cross-blocking assay such that, during the assay and in the presence of a second amino acid sequence or other binding agent of the description, the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination. The Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the description. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version. In this particular format, an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface. In addition to the example given using N-terminal His-tagged target, C-terminal His-tagged target could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).

[0168] The following generally describes an ELISA assay for determining whether an amino acid sequence or other binding agent directed against a target cross-blocks or is capable of cross-blocking as defined herein. It will be appreciated that the assay can be used with any of the amino acid sequences (or other binding agents such as polypeptides of the description) described herein. The general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules. The plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target. The amount of bound target is then measured using a reagent that is appropriate to detect the target. An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence. In the instance where the first amino acid sequence, e.g. an Ab-X, is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second amino acid sequence, i.e. Ab-Y, is then added to the ELISA plate such that the moles of Ab-Y target binding sites per well are at least 10 fold higher than the moles of Ab-X target binding sites that were used, per well, during the coating of the ELISA plate. Target is then added such that the moles of target added per well are at least 25-fold lower than the moles of Ab-X target binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti-target amino acid sequence (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y), target buffer only (i.e. without target) and target detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence buffer only (i.e. without second solution phase amino acid sequence), target and target detection reagents. The ELISA assay may be run in such a manner so as to have the positive control signal be at least 6 times the background signal. To avoid any artefacts (e.g. significantly different affinities between Ab-X and Ab-Y for the target) resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which to use as the second (competitor) amino acid sequence, the cross-blocking assay may be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution. Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal (i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti-target amino acid sequence (i.e. the positive control wells). v) An amino acid sequence is said to be "cross-reactive" for two different antigens or antigenic determinants (such as serum albumin from two different species of mammal, such as human serum albumin and cyno serum albumin) if it is specific for (as defined herein) both these different antigens or antigenic determinants. w) By binding that is "essentially independent of the pH" is generally meant herein that the association constant (K A ) of the amino acid sequence with respect to the serum protein (such as serum albumin) at the pH value(s) that occur in a cell of an animal or human body (as further described herein) is at least 5%, such as at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 60%, such as even more preferably at least 70%, such as at least 80% or 90% or more (or even more than 100%, such as more than 110%, more than 120% or even 130% or more, or even more than 150%, or even more than 200%) of the association constant (K A ) of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell. Alternatively, by binding that is "essentially independent of the pH" is generally meant herein that the k off rate (measured by Biacore) of the amino acid sequence with respect to the serum protein (such as serum albumin) at the pH value(s) that occur in a cell of an animal or human body (as e.g. further described herein, e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 60%, such as even more preferably at least 70%, such as at least 80% or 90% or more (or even more than 100%, such as more than 110%, more than 120% or even 130% or more, or even more than 150%, or even more than 200%) of the k off rate of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By "the pH value(s) that occur in a cell of an animal or human body" is meant the pH value(s) that may occur inside a cell, and in particular inside a cell that is involved in the recycling of the serum protein. In particular, by "the pH value(s) that occur in a cell of an animal or human body" is meant the pH value(s) that may occur inside a (sub)cellular compartment or vesicle that is involved in recycling of the serum protein (e.g. as a result of pinocytosis, endocytosis, transcytosis, exocytosis and phagocytosis or a similar mechanism of uptake or internalization into said cell), such as an endosome, lysosome or pinosome. x) As further described herein, the total number of amino acid residues in a Nanobody can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a Nanobody are not particularly limited as to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein; y) As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079, the amino acid residues of a Nanobody are numbered according to the general numbering for V H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to V HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195 (see for example Figure 2 of this publication), and accordingly FR1 of a Nanobody comprises the amino acid residues at positions 1-30, CDR1 of a Nanobody comprises the amino acid residues at positions 31-35, FR2 of a Nanobody comprises the amino acids at positions 36-49, CDR2 of a Nanobody comprises the amino acid residues at positions 50-65, FR3 of a Nanobody comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody comprises the amino acid residues at positions 103-113. z) The Figures, Sequence Listing and the Experimental Part / Examples are only given to further illustrate the invention.

[0169] For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the prior art cited herein, as well as to the prior art mentioned on page 59 of WO 08 / 020079 and to the list of references mentioned on pages 41-43 of the International application WO 06 / 040153.

[0170] In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as "V HH domains", in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "V H domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "V L domains").

[0171] As mentioned in the prior art referred to above, V HH domains have a number of unique structural characteristics and functional properties which make isolated V HH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring V HH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, V HH domains (which have been "designed" by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the V HH domains from the V H and V L domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a V H domain covalently linked to a V L domain).

[0172] Because of these unique properties, the use of V HH domains and Nanobodies as single antigen-binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional V H and V L domains, scFv's or conventional antibody fragments (such as Fab- or F(ab') 2 -fragments), including the advantages that are listed on pages 60 and 61 of WO 08 / 020079.

[0173] In a specific and preferred aspect, the description provides Nanobodies against OX40L, and in particular Nanobodies against OX40L from a warm-blooded animal, and more in particular Nanobodies against OX40L from a mammal, and especially Nanobodies against human OX40L; as well as proteins and / or polypeptides comprising at least one such Nanobody.

[0174] In particular, the description provides Nanobodies against OX40L, and proteins and / or polypeptides comprising the same, that have improved therapeutic and / or pharmacological properties and / or other advantageous properties (such as, for example, improved ease of preparation and / or reduced costs of goods), compared to conventional antibodies against OX40L or fragments thereof, compared to constructs that could be based on such conventional antibodies or antibody fragments (such as Fab' fragments, F(ab') 2 fragments, ScFv constructs, "diabodies" and other multispecific constructs (see for example the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep;23(9):1126-36)), and also compared to the so-called "dAb's" or similar (single) domain antibodies that may be derived from variable domains of conventional antibodies. These improved and advantageous properties will become clear from the further description herein, and for example include, without limitation, one or more of: increased affinity and / or avidity for OX40L, either in a monovalent format, in a multivalent format (for example in a bivalent format) and / or in a multispecific format (for example one of the multispecific formats described hereinbelow); better suitability for formatting in a multivalent format (for example in a bivalent format); better suitability for formatting in a multispecific format (for example one of the multispecific formats described hereinbelow); improved suitability or susceptibility for "humanizing" substitutions (as defined herein); less immunogenicity, either in a monovalent format, in a multivalent format (for example in a bivalent format) and / or in a multispecific format (for example one of the multispecific formats described hereinbelow); increased stability, either in a monovalent format, in a multivalent format (for example in a bivalent format) and / or in a multispecific format (for example one of the multispecific formats described hereinbelow); increased specificity towards OX40L, either in a monovalent format, in a multivalent format (for example in a bivalent format) and / or in a multispecific format (for example one of the multispecific formats described hereinbelow); decreased or where desired increased cross-reactivity with OX40L from different species; and / or one or more other improved properties desirable for pharmaceutical use (including prophylactic use and / or therapeutic use) and / or for diagnostic use (including but not limited to use for imaging purposes), either in a monovalent format, in a multivalent format (for example in a bivalent format) and / or in a multispecific format (for example one of the multispecific formats described hereinbelow).

[0175] As generally described herein for the amino acid sequences of the description, the Nanobodies of the description are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the description (as defined herein), which may comprise or essentially consist of one or more Nanobodies of the description and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the description may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than OX40L), so as to provide a monovalent, multivalent or multispecific polypeptide of the description, respectively, all as described herein. In particular, such a protein or polypeptide may comprise or essentially consist of one or more Nanobodies of the description and optionally one or more (other) Nanobodies (i.e. directed against other targets than OX40L), all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent or multispecific Nanobody construct, respectively, as further described herein. Such proteins or polypeptides may also be in essentially isolated form (as defined herein).

[0176] In a Nanobody of the description, the binding site for binding against OX40L is preferably formed by the CDR sequences. Optionally, a Nanobody of the description may also, and in addition to the at least one binding site for binding against OX40L, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston, Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130; and WO 06 / 07260.

[0177] As generally described herein for the amino acid sequences of the description, when a Nanobody of the description (or a polypeptide of the description comprising the same) is intended for administration to a subject (for example for therapeutic and / or diagnostic purposes as described herein), it is preferably directed against human OX40L; whereas for veterinary purposes, it is preferably directed against OX40L from the species to be treated. Also, as with the amino acid sequences of the description, a Nanobody of the description may or may not be cross-reactive (i.e. directed against OX40L from two or more species of mammal, such as against human OX40L and OX40L from at least one of the species of mammal mentioned herein).

[0178] Also, again as generally described herein for the amino acid sequences of the description, the Nanobodies of the description may generally be directed against any antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of OX40L.

[0179] As already described herein, the amino acid sequence and structure of a Nanobody can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's" (or sometimes also referred to as "FW's"), which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region 1"or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. Some preferred framework sequences and CDR's (and combinations thereof) that are present in the Nanobodies of the description are as described herein. Other suitable CDR sequences can be obtained by the methods described herein.

[0180] According to a non-limiting but preferred aspect of the description, (the CDR sequences present in) the Nanobodies of the description are such that: the Nanobodies can bind to OX40L with a dissociation constant (K D ) of 10 -5< to 10 -12< moles / liter or less, and preferably 10 -7< to 10 -12< moles / liter or less and more preferably 10 -8< to 10 -12< moles / liter (i.e. with an association constant (K A ) of 10 5< to 10 12< liter / moles or more, and preferably 10 7< to 10 12< liter / moles or more and more preferably 10 8< to 10 12< liter / moles); and / or such that: the Nanobodies can bind to OX40L with a k on -rate of between 10 2< M -1< s -1< to about 10 7< M -1< s -1< , preferably between 10 3< M -1< s -1< and 10 7< M -1< s -1< , more preferably between 10 4< M -1< s -1< and 10 7< M -1< s -1< , such as between 10 5< M -1< s -1< and 10 7< M -1< s -1< ; and / or such that they: the Nanobodies can bind to OX40L with a k off rate between 1 s -1< (t 1 / 2 =0.69 s) and 10 -6< s -1< (providing a near irreversible complex with a t 1 / 2 of multiple days), preferably between 10 -2< s -1< and 10 -6< s -1< , more preferably between 10 -3< s -1< and 10 -6< s -1< , such as between 10 -4< s -1< and 10 -6< s -1< .

[0181] Preferably, (the CDR sequences present in) the Nanobodies of the description are such that: a monovalent Nanobody of the description (or a polypeptide that contains only one Nanobody of the description) is preferably such that it will bind to OX40L with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

[0182] The affinity of the Nanobody of the description against OX40L can be determined in a manner known per se, for example using the general techniques for measuring K D . K A , k off or k on mentioned herein, as well as some of the specific assays described herein.

[0183] Some preferred IC50 values for binding of the Nanobodies of the description (and of polypeptides comprising the same) to OX40L will become clear from the further description and examples herein.

[0184] In a preferred but non-limiting aspect, the description relates to a Nanobody (as defined herein) against OX40L, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which: CDR1 is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; and / or CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; and / or CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; or any suitable fragment of such an amino acid sequence.

[0185] In particular, according to this preferred but non-limiting aspect, the description relates to a Nanobody (as defined herein) against OX40L, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which: CDR1 is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NO's: 133 to 139; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 133 to 139; and CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NO's: 147 to 153; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 147 to 153; and CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NO's: 161 to 167; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 161 to 167; or any suitable fragment of such an amino acid sequences.

[0186] As generally mentioned herein for the amino acid sequences of the description, when a Nanobody of the description contains one or more CDR1 sequences according to b) and / or c): i) any amino acid substitution in such a CDR according to b) and / or c) is preferably, and compared to the corresponding CDR according to a), a conservative amino acid substitution (as defined herein); and / or ii) the CDR according to b) and / or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to a); and / or iii) the CDR according to b) and / or c) may be a CDR that is derived from a CDR according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0187] Similarly, when a Nanobody of the description contains one or more CDR2 sequences according to e) and / or f): i) any amino acid substitution in such a CDR according to e) and / or f) is preferably, and compared to the corresponding CDR according to d), a conservative amino acid substitution (as defined herein); and / or ii) the CDR according to e) and / or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to d); and / or iii) the CDR according to e) and / or f) may be a CDR that is derived from a CDR according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0188] Also, similarly, when a Nanobody of the description contains one or more CDR3 sequences according to h) and / or i): i) any amino acid substitution in such a CDR according to h) and / or i) is preferably, and compared to the corresponding CDR according to g), a conservative amino acid substitution (as defined herein); and / or ii) the CDR according to h) and / or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to g); and / or iii) the CDR according to h) and / or i) may be a CDR that is derived from a CDR according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0189] It should be understood that the last three paragraphs generally apply to any Nanobody of the description that comprises one or more CDR1 sequences, CDR2 sequences and / or CDR3 sequences according to b), c), e), f), h) or i), respectively.

[0190] Of the Nanobodies of the description, Nanobodies comprising one or more of the CDR's explicitly listed above are particularly preferred; Nanobodies comprising two or more of the CDR's explicitly listed above are more particularly preferred; and Nanobodies comprising three of the CDR's explicitly listed above are most particularly preferred.

[0191] Some particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table B-1 below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies of the description. As will be clear to the skilled person, a combination of CDR1, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the same line in Table B-1) will usually be preferred (although the description in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table B-1). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table B-1) will usually be preferred (although the description in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table B-1, as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).

[0192] Also, in the Nanobodies of the description that comprise the combinations of CDR's mentioned in Table B-1, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which: i) any amino acid substitution in such a CDR is preferably, and compared to the corresponding CDR sequence mentioned in Table B-1, a conservative amino acid substitution (as defined herein); and / or ii) any such CDR sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR sequence mentioned in Table B-1; and / or iii) any such CDR sequence is a CDR that is derived by means of a technique for affinity maturation known per se, and in particular starting from the corresponding CDR sequence mentioned in Table B-1.

[0193] However, as will be clear to the skilled person, the (combinations of) CDR sequences, as well as (the combinations of) CDR sequences and framework sequences mentioned in Table B-1 will generally be preferred. Table B-1: Preferred combinations of CDR sequences, preferred combinations of framework sequences, and preferred combinations of framework and CDR sequences. ("ID" refers to the SEQ ID NO as used herein)IDFR1IDCDR1IDFR2IDCDR2IDFR3IDCDR3IDFR4126133LDRMG140147154161168127134TYIMG141148155162169128135SIYAKG142149156163170129136SFAMG143150157164171130137LNTMG144151158165172131138DYAIA145152159166173132139146153160167174

[0194] Thus, in the Nanobodies of the description, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1; or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% "sequence identity" (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1; and / or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

[0195] In this context, by "suitably chosen" is meant that, as applicable, a CDR1 sequence is chosen from suitable CDR1 sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the Nanobodies of the description bind to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein.

[0196] In particular, in the Nanobodies of the description, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table B-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table B-1; and / or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table B-1.

[0197] Preferably, in the Nanobodies of the description, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1; and / or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

[0198] In particular, in the Nanobodies of the description, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table B-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table B-1, respectively; and at least one of the CDR1 and CDR2 sequences present is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table B-1 or from the group of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table B-1; and / or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table B-1.

[0199] Most preferably, in the Nanobodies of the description, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1 or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1; and / or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

[0200] Even more preferably, in the Nanobodies of the description, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences, respectively, listed in Table B-1; and / or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table B-1.

[0201] In particular, in the Nanobodies of the description, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table B-1. Preferably, in this aspect, at least one and preferably both of the CDR1 and CDR2 sequences present are suitably chosen from the groups of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR1 and CDR2 sequences, respectively, listed in Table B-1; and / or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table B-1.

[0202] Even more preferably, in the Nanobodies of the description, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table B-1; and / or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table B-1.

[0203] In particular, in the Nanobodies of the description, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table B-1, and either the CDR1 sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table B-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table B-1; and / or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table B-1.

[0204] Even more preferably, in the Nanobodies of the description, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

[0205] Also, generally, the combinations of CDR's listed in Table B-1 (i.e. those mentioned on the same line in Table B-1) are preferred. Thus, it is generally preferred that, when a CDR in a Nanobody of the description is a CDR sequence mentioned in Table B-1 or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table B-1; and / or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table B-1, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table B-1 (i.e. mentioned on the same line in Table B-1) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and / or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR's mentioned in Table B-1.

[0206] Thus, by means of non-limiting examples, a Nanobody of the description can for example comprise a CDR1 sequence that has more than 80 % sequence identity with one of the CDR1 sequences mentioned in Table B-1, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table B-1 (but belonging to a different combination), and a CDR3 sequence.

[0207] Some preferred Nanobodies of the description may for example comprise: (1) a CDR1 sequence that has more than 80 % sequence identity with one of the CDR1 sequences mentioned in Table B-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table B-1 (but belonging to a different combination); and a CDR3 sequence that has more than 80 % sequence identity with one of the CDR3 sequences mentioned in Table B-1 (but belonging to a different combination); or (2) a CDR1 sequence that has more than 80 % sequence identity with one of the CDR1 sequences mentioned in Table B-1; a CDR2 sequence, and one of the CDR3 sequences listed in Table B-1; or (3) a CDR1 sequence; a CDR2 sequence that has more than 80% sequence identity with one of the CDR2 sequence listed in Table B-1; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table B-1 that belongs to the same combination as the CDR2 sequence.

[0208] Some particularly preferred Nanobodies of the description may for example comprise: (1) a CDR1 sequence that has more than 80 % sequence identity with one of the CDR1 sequences mentioned in Table B-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table B-1 that belongs to the same combination; and a CDR3 sequence that has more than 80 % sequence identity with the CDR3 sequence mentioned in Table B-1 that belongs to the same combination; (2) a CDR1 sequence; a CDR 2 listed in Table B-1 and a CDR3 sequence listed in Table B-1 (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).

[0209] Some even more preferred Nanobodies of the description may for example comprise: (1) a CDR1 sequence that has more than 80 % sequence identity with one of the CDR1 sequences mentioned in Table B-1; the CDR2 sequence listed in Table B-1 that belongs to the same combination; and a CDR3 sequence mentioned in Table B-1 that belongs to a different combination; or (2) a CDR1 sequence mentioned in Table B-1; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table B-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence listed in Table B-1 that belongs to the same or a different combination.

[0210] Particularly preferred Nanobodies of the description may for example comprise a CDR1 sequence mentioned in Table B-1, a CDR2 sequence that has more than 80 % sequence identity with the CDR2 sequence mentioned in Table B-1 that belongs to the same combination; and the CDR3 sequence mentioned in Table B-1 that belongs to the same combination.

[0211] In the most preferred Nanobodies of the description, the CDR1, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

[0212] According to another preferred, but non-limiting aspect of the description (a) CDR1 has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and / or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and / or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.

[0213] In another preferred, but non-limiting aspect, the description relates to a Nanobody in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1).

[0214] Generally, Nanobodies with the above CDR sequences may be as further described herein, and preferably have framework sequences that are also as further described herein. Thus, for example and as mentioned herein, such Nanobodies may be naturally occurring Nanobodies (from any suitable species), naturally occurring V HH sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences or Nanobodies, including but not limited to partially humanized Nanobodies or V HH sequences, fully humanized Nanobodies or V HH sequences, camelized heavy chain variable domain sequences, as well as Nanobodies that have been obtained by the techniques mentioned herein.

[0215] Thus, in one specific, but non-limiting aspect, the description relates to a humanized Nanobody, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which CDR1 to CDR3 are as defined herein and in which said humanized Nanobody comprises at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

[0216] In another preferred, but non-limiting aspect, the description relates to a Nanobody in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 179 to 185 (see Table A-1), in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

[0217] In another preferred, but non-limiting aspect, the description relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 179 to 185 (see Table A-1) or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1).

[0218] Another preferred, but non-limiting aspect of the description relates to humanized and / or sequence optimized variants of the Nanobodies of SEQ ID NO's: 179 to 185 (see Table A-1), that comprise, compared to the corresponding native V HH sequence, at least one humanizing and / or sequence optimizing substitution (as defined herein), and in particular at least one humanizing and / or sequence optimizing substitution in at least one of its framework sequences (as defined herein). Some preferred, but non-limiting examples of such humanized and / or sequence optimized variants are the humanized and / or sequence optimized Nanobodies of SEQ ID NO's: 199 to 226 (see Table A-2). Thus, the description also relates to a humanized and / or sequence optimized Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 199 to 226 (see Table A-2) or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 199 to 226 (see Table A-2) (in which amino acid sequences that are chosen from the latter group of amino acid sequences may contain a greater number or a smaller number of humanizing and / or sequence optimizing substitutions compared to the corresponding sequence of SEQ ID NO's: 199 to 226 (see Table A-2), as long as they retain at least one of the humanizing and / or sequence optimizing substitutions present in the corresponding sequence of SEQ ID NO's: 199 to 226 (see Table A-2)).

[0219] The polypeptides of the description comprise or essentially consist of at least one Nanobody of the description. Some preferred, but non-limiting examples of polypeptides of the description are given in SEQ ID NO's: 186 to 198 (see Table A-3) and SEQ ID NO's: 227 to 234 (see Table A-4).

[0220] It will be clear to the skilled person that the Nanobodies that are mentioned herein as "preferred" (or "more preferred", "even more preferred", etc.) are also preferred (or more preferred, or even more preferred, etc.) for use in the polypeptides described herein. Thus, polypeptides that comprise or essentially consist of one or more "preferred" Nanobodies of the description will generally be preferred, and polypeptides that comprise or essentially consist of one or more "more preferred" Nanobodies of the description will generally be more preferred, etc.

[0221] Generally, proteins or polypeptides that comprise or essentially consist of a single Nanobody (such as a single Nanobody of the description) will be referred to herein as "monovalent" proteins or polypeptides or as "monovalent constructs". Proteins and polypeptides that comprise or essentially consist of two or more Nanobodies (such as at least two Nanobodies of the description or at least one Nanobody of the description and at least one other Nanobody) will be referred to herein as "multivalent" proteins or polypeptides or as "multivalent constructs", and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the description. Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

[0222] According to one specific, but non-limiting aspect, a polypeptide of the description comprises or essentially consists of at least two Nanobodies of the description, such as two or three Nanobodies of the description. As further described herein, such multivalent constructs can provide certain advantages compared to a protein or polypeptide comprising or essentially consisting of a single Nanobody of the description, such as a much improved avidity for OX40L. Such multivalent constructs will be clear to the skilled person based on the disclosure herein; some preferred, but non-limiting examples of such multivalent Nanobody constructs are the constructs of SEQ ID NO's: 186 to 198 and 227 to 234, more preferably constructs of SEQ ID NO's: 186, 187, 189, 190, 227, 228, 229, 230, 231, 233; most preferred construct of SEQ ID NO's: 190, 227, 228, 231 and 233.

[0223] According to another specific, but non-limiting aspect, a polypeptide of the description comprises or essentially consists of at least one Nanobody of the description and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is preferably also a Nanobody. Such proteins or polypeptides are also referred to herein as "multispecific" proteins or polypeptides or as 'multispecific constructs", and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the description (as will become clear from the further discussion herein of some preferred, but-nonlimiting multispecific constructs). Such multispecific constructs will be clear to the skilled person based on the disclosure herein; some preferred, but non-limiting examples of such multispecific Nanobody constructs are the constructs of SEQ ID NO's: 186 to 198 and 227 to 234, more preferably constructs of SEQ ID NO's: 186, 187, 189, 190, 227, 228, 229, 230, 231, 233; most preferred construct of SEQ ID NO's: 190, 227, 228, 231 and 233.

[0224] According to yet another specific, but non-limiting aspect, a polypeptide of the description comprises or essentially consists of at least one Nanobody of the description, optionally one or more further Nanobodies, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the Nanobody of the description and / or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies of the description. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein.

[0225] It is also possible to combine two or more of the above aspects, for example to provide a trivalent bispecific construct comprising two Nanobodies of the description and one other Nanobody, and optionally one or more other amino acid sequences. Further non-limiting examples of such constructs, as well as some constructs that are particularly preferred within the context of the present description, will become clear from the further description herein.

[0226] In the above constructs, the one or more Nanobodies and / or other amino acid sequences may be directly linked to each other and / or suitably linked to each other via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

[0227] In one specific aspect of the description, a Nanobody of the description or a compound, construct or polypeptide of the description comprising at least one Nanobody of the description may have an increased half-life, compared to the corresponding amino acid sequence of the description. Some preferred, but non-limiting examples of such Nanobodies, compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise Nanobodies sequences or polypeptides of the description that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the description that comprise at least one additional binding site for binding to a serum protein (such as serum albumin, see for example EP 0 368 684 B1, page 4); or polypeptides of the description that comprise at least one Nanobody of the description that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the Nanobody of the description. Examples of polypeptides of the description that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more Nanobodies of the description are suitable linked to one or more serum proteins or fragments thereof (such as serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, Nanobodies or (single) domain antibodies that can bind to serum proteins such as serum albumin, serum immunoglobulins such as IgG, or transferrin); polypeptides in which a Nanobody of the description is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more Nanobodies of the description are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91 / 01743, WO 01 / 45746, WO 02 / 076489 and to the US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins" of Ablynx N.V. filed on December 5, 2006 (see also WO2008 / 068280).

[0228] Again, as will be clear to the skilled person, such Nanobodies, compounds, constructs or polypeptides may contain one or more additional groups, residues, moieties or binding units, such as one or more further amino acid sequences and in particular one or more additional Nanobodies (i.e. not directed against OX40L), so as to provide a tri- of multispecific Nanobody construct.

[0229] Generally, the Nanobodies of the description (or compounds, constructs or polypeptides comprising the same) with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the description per se. For example, the Nanobodies, compounds, constructs or polypeptides of the description with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the description per se.

[0230] In a preferred, but non-limiting aspect of the description, such Nanobodies, compound, constructs or polypeptides of the description exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the description may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

[0231] In another one aspect of the description, a polypeptide of the description comprises one or more (such as two or preferably one) Nanobodies of the description linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the description to cross the blood brain barrier. In particular, said one or more amino acid sequences that allow the resulting polypeptides of the description to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies, such as the Nanobodies described in WO 02 / 057445, of which FC44 (SEQ ID NO: 189 of WO 06 / 040153) and FC5 (SEQ ID NO: 190 of WO 06 / 040154) are preferred examples.

[0232] In particular, polypeptides comprising one or more Nanobodies of the description are preferably such that they: bind to OX40L with a dissociation constant (K D ) of 10 -5< to 10 -12< moles / liter or less, and preferably 10 -7< to 10 -12< moles / liter or less and more preferably 10 -8< to 10 -12< moles / liter (i.e. with an association constant (K A ) of 10 5< to 10 12< liter / moles or more, and preferably 10 7< to 10 12< liter / moles or more and more preferably 10 8< to 10 12< liter / moles); and / or such that they: bind to OX40L with a k on -rate of between 10 2< M -1< s -1< to about 10 7< M -1< s -1< , preferably between 10 3< M -1< s -1< and 10 7< M -1< s -1< , more preferably between 10 4< M -1< s -1< and 10 7< M -1< s -1< , such as between 10 5< M -1< s -1< and 10 7< M -1< s -1< ; and / or such that they: bind to OX40L with a k off rate between 1 s -1< (t 1 / 2 =0.69 s) and 10 -6< s -1< (providing a near irreversible complex with a t 1 / 2 of multiple days), preferably between 10 -2< s -1< and 10 -6< s -1< , more preferably between 10 -3< s -1< and 10 -6< s -1< , such as between 10 -4< s -1< and 10 -6< s -1< .

[0233] Preferably, a polypeptide that contains only one amino acid sequence of the description is preferably such that it will bind to OX40L with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more Nanobodies of the description may bind to OX40L with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the description.

[0234] Some preferred IC 50 values for binding of the amino acid sequences or polypeptides of the description to OX40L will become clear from the further description and examples herein.

[0235] Other polypeptides according to this preferred aspect of the description may for example be chosen from the group consisting of amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more "sequence identity" (as defined herein) with one or more of the amino acid sequences of SEQ ID NO's: 186 to 198 (see Table A-3) and SEQ ID NO's: 227 to 234 (Table A-4), in which the Nanobodies comprised within said amino acid sequences are preferably as further defined herein.

[0236] Another aspect of this description relates to a nucleic acid that encodes an amino acid sequence of the description (such as a Nanobody of the description) or a polypeptide of the description comprising the same. Again, as generally described herein for the nucleic acids of the description, such a nucleic acid may be in the form of a genetic construct, as defined herein.

[0237] In another aspect, the description relates to host or host cell that expresses or that is capable of expressing an amino acid sequence (such as a Nanobody) of the description and / or a polypeptide of the description comprising the same; and / or that contains a nucleic acid of the description. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

[0238] Another aspect of the description relates to a product or composition containing or comprising at least one amino acid sequence of the description, at least one polypeptide of the description and / or at least one nucleic acid of the description, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

[0239] The description further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

[0240] The description further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and / or treatment for diseases and disorders associated with OX40L. Some preferred but non-limiting applications and uses will become clear from the further description herein.

[0241] Other aspects, embodiments, advantages and applications of the description will also become clear from the further description hereinbelow.

[0242] Generally, it should be noted that the term Nanobody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the Nanobodies of the description can generally be obtained by any of the techniques (1) to (8) mentioned on pages 61 and 62 of WO 08 / 020079, or any other suitable technique known per se. One preferred class of Nanobodies corresponds to the V HH domains of naturally occurring heavy chain antibodies directed against OX40L. As further described herein, such V HH sequences can generally be generated or obtained by suitably immunizing a species of Camelid with OX40L (i.e. so as to raise an immune response and / or heavy chain antibodies directed against OX40L), by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B-cells), and by generating V HH sequences directed against OX40L, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and / or are further described herein.

[0243] Alternatively, such naturally occurring V HH domains against OX40L, can be obtained from naïve libraries of Camelid V HH sequences, for example by screening such a library using OX40L, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naïve V HH libraries may be used, such as V HH libraries obtained from naïve V HH libraries by techniques such as random mutagenesis and / or CDR shuffling, as for example described in WO 00 / 43507.

[0244] Thus, in another aspect, the description relates to a method for generating Nanobodies that are directed against OX40L. In one aspect, said method at least comprises the steps of: a) providing a set, collection or library of Nanobody sequences; and b) screening said set, collection or library of Nanobody sequences for Nanobody sequences that can bind to and / or have affinity for OX40L; and c) isolating the Nanobody or Nanobodies that can bind to and / or have affinity for OX40L.

[0245] In such a method, the set, collection or library of Nanobody sequences may be a naïve set, collection or library of Nanobody sequences; a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and / or a set, collection or library of Nanobody sequences that have been subjected to affinity maturation.

[0246] In a preferred aspect of this method, the set, collection or library of Nanobody sequences may be an immune set, collection or library of Nanobody sequences, and in particular an immune set, collection or library of V HH sequences, that have been derived from a species of Camelid that has been suitably immunized with OX40L or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

[0247] In the above methods, the set, collection or library of Nanobody or V HH sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) Nanobody sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03 / 054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0248] In another aspect, the method for generating Nanobody sequences comprises at least the steps of: a) providing a collection or sample of cells derived from a species of Camelid that express immunoglobulin sequences; b) screening said collection or sample of cells for (i) cells that express an immunoglobulin sequence that can bind to and / or have affinity for OX40L; and (ii) cells that express heavy chain antibodies, in which substeps (i) and (ii) can be performed essentially as a single screening step or in any suitable order as two separate screening steps, so as to provide at least one cell that expresses a heavy chain antibody that can bind to and / or has affinity for OX40L; and c) either (i) isolating from said cell the V HH sequence present in said heavy chain antibody; or (ii) isolating from said cell a nucleic acid sequence that encodes the V HH sequence present in said heavy chain antibody, followed by expressing said V HH domain.

[0249] In the method according to this aspect, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with OX40L or a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

[0250] The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05 / 19824, WO 04 / 051268 and WO 04 / 106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is made to the so-called "Nanoclone ™< " technique described in International application WO 06 / 079372 by Ablynx N.V.

[0251] In another aspect, the method for generating an amino acid sequence directed against OX40L may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode a heavy chain antibody or a Nanobody sequence that can bind to and / or has affinity for OX40L; and c) isolating said nucleic acid sequence, followed by expressing the V HH sequence present in said heavy chain antibody or by expressing said Nanobody sequence, respectively.

[0252] In such a method, the set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of heavy chain antibodies or V HH sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and / or a set, collection or library of nucleic acid sequences encoding a set, collection or library of Nanobody sequences that have been subjected to affinity maturation.

[0253] In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or V HH sequences derived from a Camelid that has been suitably immunized with OX40L or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

[0254] In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03 / 054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

[0255] As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term "screening" as used in the present description can comprise selection, screening or any suitable combination of selection and / or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10 4< , 10 5< , 10 6< , 10 7< , 10 8< or more sequences.

[0256] Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational, or semi-empirical approaches such as computer modelling techniques or biostatics or datamining techniques.

[0257] Furthermore, such a set, collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library)), or any other source of diverse sequences (as described for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the description. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism. Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.

[0258] Yet another technique for obtaining V HH sequences or Nanobody sequences directed against OX40L involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and / or heavy chain antibodies directed against OX40L), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said V HH sequences or Nanobody sequences (such as a blood sample, serum sample or sample of B-cells), and then generating V HH sequences directed against OX40L, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02 / 085945, WO 04 / 049794 and WO 06 / 008548 and Janssens et al., Proc. Natl. Acad. Sci .USA. 2006 Oct 10;103(41):15130-5 can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.

[0259] The description also relates to the V HH sequences or Nanobody sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said V HH sequence or Nanobody sequence; and of expressing or synthesizing said V HH sequence or Nanobody sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

[0260] As mentioned herein, a particularly preferred class of Nanobodies of the description comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V HH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V HH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V H domain from a conventional 4-chain antibody from a human being (e.g. indicated above), as further described on, and using the techniques mentioned on, page 63 of WO 08 / 020079. Another particularly preferred class of Nanobodies of the description comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V H domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V HH domain of a heavy chain antibody, as further described for example on, and using the techniques mentioned on, page 63 of WO 08 / 020079.

[0261] Other suitable methods and techniques for obtaining the Nanobodies of the description and / or nucleic acids encoding the same, starting from naturally occurring V H sequences or preferably V HH sequences, will be clear from the skilled person, and may for example include the techniques that are mentioned on page 64 of WO 08 / 00279. As mentioned herein, Nanobodies may in particular be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more of the framework sequences.

[0262] Thus, according to one preferred, but non-limiting aspect of the description, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising: a) an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and / or: b) an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and / or: c) an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.

[0263] Thus, in a first preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and / or in which: b) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid or a cysteine and the amino acid residue at position 44 according to the Kabat numbering is preferably E; and / or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S; and in which: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0264] In particular, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising: a) an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and / or: b) an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and / or: c) an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.

[0265] Thus, according to a preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and / or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and / or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S; and in which: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0266] In particular, a Nanobody against OX40L according to the description may have the structure: FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and / or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and / or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S; and in which: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0267] In particular, according to one preferred, but non-limiting aspect of the description, a Nanobody can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which; a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R; and a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W; a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; or in which: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W; b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q; or in which: c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q; and in whichd) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0268] Thus, in another preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and in which: a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R; and in which: a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W; and in which a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; and in which: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0269] In another preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and in which: b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and in which: b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W; and in which: b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q; and in which: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0270] In another preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and in which: c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and in which: c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and in which: c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q; and in which: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0271] Two particularly preferred, but non-limiting groups of the Nanobodies of the description are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to (b-1) to (b-4) above; according to (c) above; and / or according to (c-1) to (c-4) above, in which either: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as described herein) and the amino acid residue at position 108 is Q; or in which: ii) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence as described) and the amino acid residue at position 108 is Q or L, and is preferably Q.

[0272] Thus, in another preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q; and in which: ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0273] In another preferred, but non-limiting aspect, a Nanobody of the description may have the structure FR1- CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q; and in which: ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0274] In the Nanobodies of the description in which the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE, the amino acid residue at position 37 is most preferably F. In the Nanobodies of the description in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW, the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.

[0275] Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies of the description can generally be classified on the basis of the following three groups: i) The "GLEW-group": Nanobodies with the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering and Q at position 108 according to the Kabat numbering. As further described herein, Nanobodies within this group usually have a V at position 37, and can have a W, P, R or S at position 103, and preferably have a W at position 103. The GLEW group also comprises some GLEW-like sequences such as those mentioned in Table B-2 below. More generally, and without limitation, Nanobodies belonging to the GLEW-group can be defined as Nanobodies with a G at position 44 and / or with a W at position 47, in which position 46 is usually E and in which preferably position 45 is not a charged amino acid residue and not cysteine; ii) The "KERE-group": Nanobodies with the amino acid sequence KERE or KQRE (or another KERE-like sequence) at positions 43-46 according to the Kabat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein, Nanobodies within this group usually have a F at position 37, an L or F at position 47; and can have a W, P, R or S at position 103, and preferably have a W at position 103. More generally, and without limitation, Nanobodies belonging to the KERE-group can be defined as Nanobodies with a K, Q or R at position 44 (usually K) in which position 45 is a charged amino acid residue or cysteine, and position 47 is as further defined herein; iii) The "103 P, R, S-group": Nanobodies with a P, R or S at position 103. These Nanobodies can have either the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering or the amino acid sequence KERE or KQRE at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.

[0276] Also, where appropriate, Nanobodies may belong to (i.e. have characteristics of) two or more of these classes. For example, one specifically preferred group of Nanobodies has GLEW or a GLEW-like sequence at positions 44-47; P,R or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).

[0277] More generally, it should be noted that the definitions referred to above describe and apply to Nanobodies in the form of a native (i.e. non-humanized) V HH sequence, and that humanized variants of these Nanobodies may contain other amino acid residues than those indicated above (i.e. one or more humanizing substitutions as defined herein). For example, and without limitation, in some humanized Nanobodies of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L. As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V HH sequence with the corresponding framework sequence of one or more closely related human V H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V HH sequence (in any manner known per se, as further described herein) and the resulting humanized V HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and / or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody may be partially humanized or fully humanized.

[0278] Thus, in another preferred, but non-limiting aspect, a Nanobody of the description may be a Nanobody belonging to the GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0279] In another preferred, but non-limiting aspect, a Nanobody of the description may be a Nanobody belonging to the KERE-group (as defined herein), and CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0280] Thus, in another preferred, but non-limiting aspect, a Nanobody of the description may be a Nanobody belonging to the 103 P, R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0281] Also, more generally and in addition to the 108Q, 43E / 44R and 103 P,R,S residues mentioned above, the Nanobodies of the description can contain, at one or more positions that in a conventional V H domain would form (part of) the V H / V L interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V H sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2 on page 48 of the International application WO 08 / 020079). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table B-2 below; as well as the substitutions that are described in the International Application WO 00 / 29004 for so-called "microbodies", e.g. so as to obtain a Nanobody with Q at position 108 in combination with KLEW at positions 44-47. Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.

[0282] In one aspect of the Nanobodies of the description, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.

[0283] Also, in one aspect of the Nanobodies of the description, the amino acid residue at position 83 is chosen from the group consisting of R, K, N, E, G, I, T and Q; and is most preferably either K or E (for Nanobodies corresponding to naturally occurring V HH domains) or R (for "humanized" Nanobodies, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one aspect, and is most preferably P (for Nanobodies corresponding to naturally occurring V HH domains) or R (for "humanized" Nanobodies, as described herein).

[0284] Furthermore, in one aspect of the Nanobodies of the description, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.

[0285] Collectively, the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies are as mentioned above, will also be referred to herein as the "Hallmark Residues". The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human V H domain, V H 3, are summarized in Table B-2.

[0286] Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring V HH domains are mentioned in Table B-3. For comparison, the corresponding amino acid residues of the human V H 3 called DP-47 have been indicated in italics. Table B-2: Hallmark Residues in Nanobodies PositionHuman V H 3Hallmark Residues11L, V; predominantly LL, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L37V, I, F; usually VF (1)< , Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F (1)< or Y44 (8)< GE (3)< , Q (3)< , G (2)< , D, A, K, R, L, P, S, V, H, T, N, W, M, I;preferably G (2)< , E (3)< or Q (3)< ;most preferably G (2)< or Q (3)< .45 (8)< LL (2)< , R (3)< , P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L (2)< or R (3)< 47 (8)< W, YF (1)< , L (1)< or W (2)< G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W (2)< , L (1)< or F (1)< 83R or K; usually RR, K (5)< , T, E (5)< , Q, N, S, I, V, G, M, L, A, D, Y, H; preferably K or R; most preferably K84A, T, D; predominantly AP (5)< , S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; preferably P103WW (4)< , R (6)< , G, S, K, A, M, Y, L, F, T, N, V, Q, P (6)< , E, C; preferably W104GG, A, S, T, D, P, N, E, C, L; preferably G108L, M or T; predominantly LQ, L (7)< , R, P, E, K, S, T, M, A, H; preferably Q or L (7)< Notes:(1)In particular, but not exclusively, in combination with KERE or KQRE at positions 43-46.(2)Usually as GLEW at positions 44-47.(3)Usually as KERE or KQRE at positions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47. Alternatively, also sequences such as TERE (for example TEREL), TQRE (for example TQREL), KECE (for example KECEL or KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible. Some other possible, but less preferred sequences include for example DECKL and NVCEL.(4)With both GLEW at positions 44-47 and KERE or KQRE at positions 43-46.(5)Often as KP or EP at positions 83-84 of naturally occurring V HH domains.(6)In particular, but not exclusively, in combination with GLEW at positions 44-47.(7)With the proviso that when positions 44-47 are GLEW, position 108 is always Q in (non-humanized) V HH sequences that also contain a W at 103.(8)The GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW. Table B-3: Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies. For humanization of these combinations, reference is made to the specification.11 37 44 45 47 83 84 103 104 108 DP-47 (human)MVGLWRAWGL"KERE" groupLFERLKPWGQLFERFEPWGQLFERFKPWGQLYQRLKPWGQLFLRVKPQGQLFQRLKPWGQLFERFKPWGQ"GLEW" groupLVGLWKSWGQMVGLWKPRGQ

[0287] In the Nanobodies, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the Kabat numbering) of a naturally occurring V HH domain.

[0288] Such amino acid residues will be clear to the skilled person. Tables B-4 to B-7 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 of naturally occurring V HH domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring V HH domain (and which is the most preferred amino acid residue for said position in a Nanobody) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V HH domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDR1).

[0289] In Tables B-4 - B-7, some of the non-limiting residues that can be present at each position of a human V H 3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human V H 3 domain is indicated in bold; and other preferred amino acid residues have been underlined.

[0290] For reference only, Tables B-4-B-7 also contain data on the V HH entropy ("V HH Ent.") and V HH variability ("V HH Var.") at each amino acid position for a representative sample of 7732 V HH sequences (including a.o. data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the V HH entropy and the V HH variability provide a measure for the variability and degree of conservation of amino acid residues between the 7732 V HH sequences analyzed: low values (i.e. <1, such as <0.5) indicate that an amino acid residue is highly conserved between the V HH sequences (i.e. little variability). For example, the G at position 9 and the W at position 36 have values for the V HH entropy of 0.01 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 36 is W in all 7732 sequences analysed), whereas for residues that form part of the CDR's generally values of 1.5 or more are found (data not shown). Note that the data represented below support the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR's (although the description is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et al., PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003). Table B-4: Non-limiting examples of amino acid residues in FR1 (for the footnotes, see the footnotes to Table B-2) Pos. Amino acid residue(s): V HH Ent.V HH Var.Human V H 3Camelid V HH 's1E, QE, Q, K, D, A, G, R0,4752V V, M, A, E, L0,0413Q Q, K, P, H, F, R0,0414L L , M, Q, P, R, F, V0,0215V, LV, Q, M, E, A, L, P, K, R0,3536E E, A, Q, D, K, H0,2157S, TS, F, L, W, T0,0528G, RG, R, E, V0,0419G G, R, V, A0,01110G, VG, D, R, S, K, E, A, Q, N, T, V0,22411Hallmark residue: L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L0,35412V, IV, A, L, M, E, G, T0,11213Q, K, RQ , L, R, H, P, E, K, T, S, V, D, G, A, N, M0,46314P A, P, T, V, S, D, F, N, I, E, L, R, G, Y, Q, H0,92515G G, E0116G, RG , D, E, A, S, N, V, R, K, T, P, C, L0,47417S S, F, P, Y, T, A, C, R, N0,14218L L, V, R, M, P, Q, S, A, T, K, H0,06119R, KR , T, K, S, N, G, A, I, L, Q, F, E, V, M0,36420L L , F, V, I, P, H, S0,18321S S, A, T, P, F, V, H, D, R, L, I, G0,13322C C , W0123A, TA, V, T, E, S, L, G, I, K, Q, R, D, F, N, P, M0,88524A A, D, V, T, H, Y, P, G, S, F, L, I, N, Q, E, R0,78925S S , P, T, A, F, L, N, Y, R, H, D, V, I, W, G, K, Q, C0,2226G G, E, R, V, T, A, S, K, D, L, I, Q, N, F, Y, M, W, P, H0,45627F R , F, S, P, L, G, I, N, T, D, H, V, E, A, Y, K, M, Q, W, C1,891228T T, I, S, A, P, F, D, N, V, R, M, L, G, Y, K, E, H, W, Q1,291229F, VF, L, S, V, I, A, W, Y, G, D, R, T, P, N, E, M, H, Q, K, C1,231130S, D, GS, D, N, G, R, T, A, E, I, Y, K, V, H, L, F, W, M, P, C, Q1,5512 Table B-5: Non-limiting examples of amino acid residues in FR2 (for the footnotes, see the footnotes to Table B-2) Pos. Amino acid residue(s): V HH Ent.V HH Var.Human V H 3Camelid V HH 's36W W 0137Hallmark residue: F (1)< , Y, V, L, A, H, S, I, W, C, N, G, D, T, P , preferably F (1)< or Y1,1738R R, H, C, P, Y, L, V0,01139Q Q, E, R, H, L, A, S, K, P, V, T, D0,22340A A, V, T, P, G, S, D, I, L, R, N, F, Y, C, E, H0,55641P, S, TP, S, A, L, T, Q, R, V, D, G, I, H0,18342G G , E, A, R, D, V, W, T, Q, K, L, N, H, M0,1243K K, N, Q, E, R, T, L, S, M, D, G, A, V, H, I, F, P0,45744Hallmark residue: E (3)< , Q (3)< , G (2)< , D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G (2)< , E (3)< or Q (3)< ; most preferably G (2)< or Q (3)< .1,11445Hallmark residue: L (2)< , R (3)< , P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L (2)< or R (3)< 0,56346E, VE, D, A, Q, V, M, K, T, G, R, S, N, I, L, F0,42447Hallmark residue: F (1)< , L (1)< or W (2)< G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W (2)< , L (1)< or F (1)< 1,641148V V, I, L, A, T, Q, F, M, G, E, R0,35549S, A, GA, S, G, T, V, L, C, I, F, P, E, Y, M, D, R0,895 Table B-6: Non-limiting examples of amino acid residues in FR3 (for the footnotes, see the footnotes to Table B-2) Pos. Amino acid residue(s): V HH Ent.V HH Var.Human V H 3Camelid V HH 's66R R 0167F F , S, L, V, I, C, A, Y, M, G0,1168T T, A, S, I, F, V, P, N, G, R, K, M, D, L, W, Q0,34469I I, V, M, T, L, A, F, P, S, G, N0,5570S S, T, A, F, P, V, Y, L, D, G, N, H, W, E, C0,22471R R , S, K, G, T, I, W, A, N, V, E, L, M, F, D, Q, C0,61772D, ED, N, E, G, V, A, H, L, S, T, I, Q, F, P, Y, R0,34473N, D, GN, D, S, K, I, Y, G, T, H, R, A, V, F, L, E, M, P, C0,65974A, SA, T, V, S, F, G, D, P, N, I, R, L, Y, H, E, Q, K, W, M0,8875K K, N, E, R, Q, A, G, T, M, S, L, D, V, W, Y, I0,71676N, SN , K, S, R, D, T, H, G, E, A, Y, I, M, Q, L, W, P, F, V0,66777S, T, IT, A, M, S, R, I, V, L, P, E, N, K, G, W, Q0,72778L, AV, L, A, M, I, G, T, F, W, Q, S, E, N, H1,11679Y , HY , F, D, S, H, N, T, A, L, W, V, C, G, E, I, P, R0,68880L L, M, V, P, F0,05281Q Q, E, R, H, L, D, T, G, K, P, A, I, S, N, Y, V, M0,38482M M, I, L, V, A, T, S, K0,12382aN, GN, S, D, T, E, H, K, I, A, G, R, Y, L, V, F, Q0,77582bS S, N, T, G, H, D, R, A, K, I, M, V, F, E, P, Y, C, L0,72882cL L, V, M, P, A, T, G0,08283Hallmark residue: R, K (5)< , T, E (5)< , Q, N, S, I, V, G, M, L, A, D, Y, H; preferably K or R; most preferably K0,66684Hallmark residue: P (5)< , S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; preferably P0,85785E, GE, D, G, A, Q, V, S, N, K, T, R, L0,27386D D, E, G, N0,02187T , MT , S, A, M, R, P, K, E0,15388A A, G, S, D, N, T, P, V0,23289V, LV, I, L, E, A, R, T, D, F, M, N, S, K, G, Q, H0,71790Y Y , H, F, N0191Y, HY, F, R, S, H, T, I, V, L, N, D, C, Q, W, A, E, M0,6792C C, R, P0193A, K, TA, N, T, K, G, V, R, Y, S, H, W, L, F, Q, M, I, E, C, D1,331094K, R, TA , K, V, T, R, L, G, S, D, Q, I, M, F, Y, N, E, H, P, C, W1,5512 Table B-7: Non-limiting examples of amino acid residues in FR4 (for the footnotes, see the footnotes to Table B-2) Pos. Amino acid residue(s): V HH Ent.V HH Var.Human V H 3Camelid V HH 's103Hallmark residue: W (4)< , R (6)< , G, S, K, A, M, Y, L, F, T, N, V, Q, P (6)< , E, C; preferably W0,546104Hallmark residue: G, A, S, T, D, P, N, E, C, L; preferably G0,133105Q, RQ , K, H, R, P, E, L, T, N, S, V, A, M, G0,525106G G, R, E01107T T, Q, I, A, S, N, R, V, D0,243108Hallmark residue: Q, L (7)< , R, P, E, K, S, T, M, A, H; preferably Q or L (7)< 0,34109V V, I, L01110T T, S, N, A, I, F0,011111V V, I, A0,011112S S , T, P, F, A0,011113S S , T, A, L, P, F, E, V0,041

[0291] Thus, in another preferred, but not limiting aspect, a Nanobody of the description can be defined as an amino acid sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2; and in which: ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0292] The above Nanobodies may for example be V HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

[0293] In particular, a Nanobody of the description can be an amino acid sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) (preferably) one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 (it being understood that V HH sequences will contain one or more Hallmark residues; and that partially humanized Nanobodies will usually, and preferably, [still] contain one or more Hallmark residues [although it is also within the scope of the description to provide - where suitable in accordance with the description - partially humanized Nanobodies in which all Hallmark residues, but not one or more of the other amino acid residues, have been humanized]; and that in fully humanized Nanobodies, where suitable in accordance with the description, all amino acid residues at the positions of the Hallmark residues will be amino acid residues that occur in a human V H 3 sequence. As will be clear to the skilled person based on the disclosure herein that such V HH sequences, such partially humanized Nanobodies with at least one Hallmark residue, such partially humanized Nanobodies without Hallmark residues and such fully humanized Nanobodies all form aspects of this description); and in which: ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded; and in which: iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0294] The above Nanobodies may for example be V HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein. Table B-8: Representative amino acid sequences for Nanobodies of the KERE, GLEW and P,R,S 103 group. The CDR's are indicated with XXXXXKERE sequence no. 1SEQ ID NO:1KERE sequence no. 2SEQ ID NO:2KERE sequence no. 3SEQ ID NO:3KERE sequence no. 4SEQ ID NO:4KERE sequence no. 5SEQ ID NO:5KERE sequence no. 6SEQ ID NO:6KERE sequence no. 7SEQ ID NO:7KERE sequence no. 8SEQ ID NO:8KERE sequence no. 9SEQ ID NO:9KERE sequence no. 10SEQ ID NO:10KERE sequence no. 11SEQ ID NO:11KERE sequence no. 12SEQ ID NO:12KERE sequence no. 13SEQ ID NO:13KERE sequence no. 14SEQ ID NO:14KERE sequence no. 15SEQ ID NO:15KERE sequence no. 16SEQ ID NO:16GLEW sequence no. 1SEQ ID NO:17GLEW sequence no. 2SEQ ID NO:18GLEW sequence no. 3SEQ ID NO:19P,R,S 103 sequence no. 1SEQ ID NO:20P,R,S 103 sequence no. 2SEQ ID NO:21P,R,S 103 sequence no. 3SEQ ID NO:22

[0295] In particular, a Nanobody of the description of the KERE group can be an amino acid sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which: i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and in which: ii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-9: Representative FW1 sequences for Nanobodies of the KERE-group. KERE FW1 sequence no. 1SEQ ID NO:23QVQRVESGGGLVQAGGSLRLSCAASGRTSSKERE FW1 sequence no. 2SEQ ID NO:24QVQLVESGGGLVQTGDSLSLSCSASGRTFSKERE FW1 sequence no. 3SEQ ID NO:25QVKLEESGGGLVQAGDSLRLSCAATGRAFGKERE FW1 sequence no. 4SEQ ID NO:26AVQLVESGGGLVQPGESLGLSCVASGRDFVKERE FW1 sequence no. 5SEQ ID NO:27EVQLVESGGGLVQAGGSLRLSCEVLGRTAGKERE FW1 sequence no. 6SEQ ID NO:28QVQLVESGGGWVQPGGSLRLSCAASETILSKERE FW1 sequence no. 7SEQ ID NO:29QVQLVESGGGTVQPGGSLNLSCVASGNTFNKERE FW1 sequence no. 8SEQ ID NO:30EVQLVESGGGLAQPGGSLQLSCSAPGFTLDKERE FW1 sequence no. 9SEQ ID NO:31AQELEESGGGLVQAGGSLRLSCAASGRTFN and in which: iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-10: Representative FW2 sequences for Nanobodies of the KERE-group. KERE FW2 sequence no. 1SEQ ID NO:41WFRQAPGKEREFVAKERE FW2 sequence no. 2SEQ ID NO:42WFRQTPGREREFVAKERE FW2 sequence no. 3SEQ ID NO:43WYRQAPGKQREMVAKERE FW2 sequence no. 4SEQ ID NO:44WYRQGPGKQRELVAKERE FW2 sequence no. 5SEQ ID NO:45WIRQAPGKEREGVSKERE FW2 sequence no. 6SEQ ID NO:46WFREAPGKEREGISKERE FW2 sequence no. 7SEQ ID NO:47WYRQAPGKERDLVAKERE FW2 sequence no. 8SEQ ID NO:48WFRQAPGKQREEVSKERE FW2 sequence no. 9SEQ ID NO:49WFRQPPGKVREFVG and in which: iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-11: Representative FW3 sequences for Nanobodies of the KERE-group. KERE FW3 sequence no. 1SEQ ID NO:50RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFKERE FW3 sequence no. 2SEQ ID NO:51RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAAKERE FW3 sequence no. 3SEQ ID NO:52RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAAKERE FW3 sequence no. 4SEQ ID NO:53RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAAKERE FW3 sequence no. 5SEQ ID NO:54RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAAKERE FW3 sequence no. 6SEQ ID NO:55RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAAKERE FW3 sequence no. 7SEQ ID NO:56RFTISKDSGKNTVYLQMTSLKPEDTAVYYCATKERE FW3 sequence no. 8SEQ ID NO:57RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAKERE FW3 sequence no. 9SEQ ID NO:58RFTISRENDKSTVYLQLNSLKPEDTAVYYCAAKERE FW3 sequence no. 10SEQ ID NO:59RFTISRDYAGNTAYLQMNSLKPEDTGVYYCAT and in which: v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-12: Representative FW4 sequences for Nanobodies of the KERE-group. KERE FW4 sequence no. 1SEQ ID NO:60WGQGTQVTVSSKERE FW4 sequence no. 2SEQ ID NO:61WGKGTLVTVSSKERE FW4 sequence no. 3SEQ ID NO:62RGQGTRVTVSSKERE FW4 sequence no. 4SEQ ID NO:63WGLGTQVTISS and in which: vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0296] In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V HH sequences or partially humanized Nanobodies).

[0297] Also, the above Nanobodies may for example be V HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

[0298] With regard to framework 1, it will be clear to the skilled person that, when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e. amino acid residues 1-4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.

[0299] Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR's), it has been found by analysis of a database of more than 1000 V HH sequences that the positions 27 to 30 have a variability (expressed in terms of V HH entropy and V HH variability - see Tables B-4 to B-7) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded.

[0300] In view of this, a Nanobody of the KERE class may be an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which: i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and in which: ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-13: Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE-group. KERE FW1 sequence no. 10SEQ ID NO:32VESGGGLVQPGGSLRLSCAASGKERE FW1 sequence no. 11SEQ ID NO:33VDSGGGLVQAGDSLKLSCALTGKERE FW1 sequence no. 12SEQ ID NO:34VDSGGGLVQAGDSLRLSCAASGKERE FW1 sequence no. 13SEQ ID NO:35VDSGGGLVEAGGSLRLSCQVSEKERE FW1 sequence no. 14SEQ ID NO:36QDSGGGSVQAGGSLKLSCAASGKERE FW1 sequence no. 15SEQ ID NO:37VQSGGRLVQAGDSLRLSCAASEKERE FW1 sequence no. 16SEQ ID NO:38VESGGTLVQSGDSLKLSCASSTKERE FW1 sequence no. 17SEQ ID NO:39MESGGDSVQSGGSLTLSCVASGKERE FW1 sequence no. 18SEQ ID NO:40QASGGGLVQAGGSLRLSCSASV and in which: iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies of the KERE-class; and in which: iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0301] The above Nanobodies may for example be V HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

[0302] A Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which i) preferably, when the Nanobody of the GLEW-class is a non-humanized Nanobody, the amino acid residue in position 108 is Q; ii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-14: Representative FW1 sequences for Nanobodies of the GLEW-group. GLEW FW1 sequence no. 1SEQ ID NO:64QVQLVESGGGLVQPGGSLRLSCAASGFTFSGLEW FW1 sequence no. 2SEQ ID NO:65EVHLVESGGGLVRPGGSLRLSCAAFGFIFKGLEW FW1 sequence no. 3SEQ ID NO:66QVKLEESGGGLAQPGGSLRLSCVASGFTFSGLEW FW1 sequence no. 4SEQ ID NO:67EVQLVESGGGLVQPGGSLRLSCVCVSSGCTGLEW FW1 sequence no. 5SEQ ID NO:68EVQLVESGGGLALPGGSLTLSCVFSGSTFS and in which: iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-15: Representative FW2 sequences for Nanobodies of the GLEW-group. GLEW FW2 sequence no. 1SEQ ID NO:72WVRQAPGKVLEWVSGLEW FW2 sequence no. 2SEQ ID NO:73WVRRPPGKGLEWVSGLEW FW2 sequence no. 3SEQ ID NO:74WVRQAPGMGLEWVSGLEW FW2 sequence no. 4SEQ ID NO:75WVRQAPGKEPEWVSGLEW FW2 sequence no. 5SEQ ID NO:76WVRQAPGKDQEWVSGLEW FW2 sequence no. 6SEQ ID NO:77WVRQAPGKAEEWVSGLEW FW2 sequence no. 7SEQ ID NO:78WVRQAPGKGLEWVAGLEW FW2 sequence no. 8SEQ ID NO:79WVRQAPGRATEWVS and in which: iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-16: Representative FW3 sequences for Nanobodies of the GLEW-group. GLEW FW3 sequence no. 1SEQ ID NO:80RFTISRDNAKNTLYLQM NSLKPEDT A VYYCVKGLEW FW3 sequence no. 2SEQ ID NO:81RFTISRDNARNTLYLQMDSLIPEDTALYYCARGLEW FW3 sequence no. 3SEQ ID NO:82RFTSSRDNAKSTLYLQM NDLKPEDTALYYCARGLEW FW3 sequence no. 4SEQ ID NO:83RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQRGLEW FW3 sequence no. 5SEQ ID NO:84RFTASRDNAKNTLYLQM NSLKSEDTARYYCARGLEW FW3 sequence no. 6SEQ ID NO:85RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGR and in which: v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-17: Representative FW4 sequences for Nanobodies of the GLEW-group. GLEW FW4 sequence no. 1SEQ ID NO:86GSQGTQVTVSSGLEW FW4 sequence no. 2SEQ ID NO:87LRGGTQVTVSSGLEW FW4 sequence no. 3SEQ ID NO:88RGQGTLVTVSSGLEW FW4 sequence no. 4SEQ ID NO:89RSRGIQVTVSSGLEW FW4 sequence no. 5SEQ ID NO:90WGKGTQVTVSSGLEW FW4 sequence no. 6SEQ ID NO:91WGQGTQVTVSS and in which: vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0303] In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V HH sequences or partially humanized Nanobodies).

[0304] With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

[0305] In view of this, a Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which: i) preferably, when the Nanobody of the GLEW-class is a non-humanized Nanobody, the amino acid residue in position 108 is Q; and in which: ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-18: Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE-group. GLEW FW1 sequence no. 6SEQ ID NO:69VESGGGLVQPGGSLRLSCAASGGLEW FW1 sequence no. 7SEQ ID NO:70EESGGGLAQPGGSLRLSCVASGGLEW FW1 sequence no. 8SEQ ID NO:71VESGGGLALPGGSLTLSCVFSG and in which: iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies of the GLEW-class; and in which: iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0306] The above Nanobodies may for example be V HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein. In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V HH sequences or partially humanized Nanobodies).

[0307] A Nanobody of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which i) the amino acid residue at position 103 according to the Kabat numbering is different from W; and in which: ii) preferably the amino acid residue at position 103 according to the Kabat numbering is P, R or S, and more preferably R; and in which:iii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-19: Representative FW1 sequences for Nanobodies of the P,R,S 103-group. P,R,S 103 FW1 sequence no. 1SEQ ID NO:92AVQLVESGGGLVQAGGSLRLSCAASGRTFSP,R,S 103 FW1 sequence no. 2SEQ ID NO:93QVQLQESGGG MVQPGGS LR LSCAASG F D FGP,R,S 103 FW1 sequence no. 3SEQ ID NO:94EVHLVESGGGLVRPGGSLRLSCAAFGFIFKP,R,S 103 FW1 sequence no. 4SEQ ID NO:95QVQLAESGGGLVQPGGSLKLSCAASRTIVSP,R,S 103 FW1 sequence no. 5SEQ ID NO:96QEHLVESGGGLVDIGGSLRLSCAASERIFSP,R,S 103 FW1 sequence no. 6SEQ ID NO:97QVKLEESGGGLAQPGGSLRLSCVASGFTFSP,R,S 103 FW1 sequence no. 7SEQ ID NO:98EVQLVESGGGLVQPGGSLRLSCVCVSSGCTP,R,S 103 FW1 sequence no. 8SEQ ID NO:99EVQLVESGGGLALPGGSLTLSCVFSGSTFS and in which iv) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-20: Representative FW2 sequences for Nanobodies of the P,R,S 103-group. P,R,S 103 FW2 sequence no. 1SEQ ID NO:102WFRQAPGKEREFVAP,R,S 103 FW2 sequence no. 2SEQ ID NO:103WVRQAPGKVLEWVSP,R,S 103 FW2 sequence no. 3SEQ ID NO:104WVRRPPGKGLEWVSP,R,S 103 FW2 sequence no. 4SEQ ID NO:105WIRQAPGKEREGVSP,R,S 103 FW2 sequence no. 5SEQ ID NO:106WVRQYPGKEPEWVSP,R,S 103 FW2 sequence no. 6SEQ ID NO:107WFRQPPGKEHEFVAP,R,S 103 FW2 sequence no. 7SEQ ID NO:108WYRQAPGKRTELVAP,R,S 103 FW2 sequence no. 8SEQ ID NO:109WLRQAPGQGLEWVSP,R,S 103 FW2 sequence no. 9SEQ ID NO:110WLRQTPGKGLEWVGP,R,S 103 FW2 sequence no. 10SEQ ID NO:111WVRQAPGKAEEFVS and in which: v) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-21: Representative FW3 sequences for Nanobodies of the P,R,S 103-group. P,R,S 103 FW3 sequence no. 1SEQ ID NO:112RFTISRDNAKNTVYLQMNSLKPEDT A VYYCAAP,R,S 103 FW3 sequence no. 2SEQ ID NO:113RFTISRDNARNTLYLQMDSLIPEDTALYYCARP,R,S 103 FW3 sequence no. 3SEQ ID NO:114RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGAP,R,S 103 FW3 sequence no. 4SEQ ID NO:115RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAAP,R,S 103 FW3 sequence no. 5SEQ ID NO:116RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRRP,R,S 103 FW3 sequence no. 6SEQ ID NO:117RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNLP,R,S 103 FW3 sequence no. 7SEQ ID NO:118RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRP,R,S 103 FW3 sequence no. 8SEQ ID NO:119RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV and in which: vi) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-22: Representative FW4 sequences for Nanobodies of the P,R,S 103-group. P,R,S 103 FW4 sequence no. 1SEQ ID NO:120RGQGTQVTVSSP,R,S 103 FW4 sequence no. 2SEQ ID NO:121LRGGTQVTVSSP,R,S 103 FW4 sequence no. 3SEQ ID NO:122GNKGTLVTVSSP,R,S 103 FW4 sequence no. 4SEQ ID NO:123SSPGTQVTVSSP,R,S 103 FW4 sequence no. 5SEQ ID NO:124SSQGTLVTVSSP,R,S 103 FW4 sequence no. 6SEQ ID NO:125RSRGIQVTVSS and in which: vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0308] In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V HH sequences or partially humanized Nanobodies).

[0309] With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

[0310] In view of this, a Nanobody of the P,R,S 103 class may be an amino acid sequence that is comprised of four framework regions / sequences interrupted by three complementarity determining regions / sequences, in which: i) the amino acid residue at position 103 according to the Kabat numbering is different from W; and in which: ii) preferably the amino acid residue at position 103 according to the Kabat numbering is P, R or S, and more preferably R; and in which: iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-23: Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the P,R,S 103-group. P,R,S 103 FW1 sequence no. 9SEQ ID NO:100VESGGGLVQAGGSLRLSCAASGP,R,S 103 FW1 sequence no. 10SEQ ID NO:101AESGGGLVQPGGSLKLSCAASR and in which: iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies of the P,R,S 103 class; and in which: v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

[0311] The above Nanobodies may for example be V HH sequences or may be humanized Nanobodies. When the above Nanobody sequences are V HH sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

[0312] In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V HH sequences or partially humanized Nanobodies).

[0313] In another preferred, but non-limiting aspect, the description relates to a Nanobody as described above, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1). This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 179 to 185 (see Table A-1), in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

[0314] As already mentioned herein, another preferred but non-limiting aspect of the description relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 179 to 185 (see Table A-1) or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 179 to 185 (see Table A-1).

[0315] Also, in the above Nanobodies: i) any amino acid substitution (when it is not a humanizing substitution as defined herein) is preferably, and compared to the corresponding amino acid sequence of SEQ ID NO's: 179 to 185 (see Table A-1), a conservative amino acid substitution, (as defined herein); and / or: ii) its amino acid sequence preferably contains either only amino acid substitutions, or otherwise preferably no more than 5, preferably no more than 3, and more preferably only 1 or 2 amino acid deletions or insertions, compared to the corresponding amino acid sequence of SEQ ID NO's: 179 to 185 (see Table A-1); and / or iii) the CDR's may be CDR's that are derived by means of affinity maturation, for example starting from the CDR's of to the corresponding amino acid sequence of SEQ ID NO's: 179 to 185 (see Table A-1).

[0316] Preferably, the CDR sequences and FR sequences in the Nanobodies of the description are such that the Nanobodies of the description (and polypeptides of the description comprising the same): bind to OX40L with a dissociation constant (K D ) of 10 -5< to 10 -12< moles / liter or less, and preferably 10 -7< to 10 -12< moles / liter or less and more preferably 10 -8< to 10 -12< moles / liter (i.e. with an association constant (K A ) of 10 5< to 10 12< liter / moles or more, and preferably 10' to 10 12< liter / moles or more and more preferably 10 8< to 10 12< liter / moles); and / or such that they: bind to OX40L with a k on -rate of between 10 2< M -1< s -1< to about 10 7< M -1< s -1< , preferably between 10 3< M -1< s -1< and 10 7< M -1< s -1< , more preferably between 10 4< M -1< s -1< and 10 7< M -1< s -1< , such as between 10 5< M -1< s -1< and 10 7< M -1< s -1< ; and / or such that they: bind to OX40L with a k off rate between 1 s -1< (t 1 / 2 =0.69 s) and 10 -6< s -1< (providing a near irreversible complex with a t 1 / 2 of multiple days), preferably between 10 -2< s -1< and 10 -6< s -1< , more preferably between 10 -3< s -1< and 10 -6< s -1< , such as between 10 -4< s -1< and 10 -6< s -1< .

[0317] Preferably, CDR sequences and FR sequences present in the Nanobodies of the description are such that the Nanobodies of the description will bind to OX40L with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

[0318] According to one non-limiting aspect of the description, a Nanobody may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human V H domain, and in particular compared to the corresponding framework region of DP-47. More specifically, according to one non-limiting aspect of the description, a Nanobody may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and / or 45) compared to the corresponding framework region of a naturally occurring human V H domain, and in particular compared to the corresponding framework region of DP-47. Usually, a Nanobody will have at least one such amino acid difference with a naturally occurring V H domain in at least one of FR2 and / or FR4, and in particular at at least one of the Hallmark residues in FR2 and / or FR4 (again, including those at positions 108, 103 and / or 45).

[0319] Also, a humanized Nanobody of the description may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring V HH domain. More specifically, according to one non-limiting aspect of the description, a humanized Nanobody may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and / or 45) compared to the corresponding framework region of a naturally occurring V HH domain. Usually, a humanized Nanobody will have at least one such amino acid difference with a naturally occurring V HH domain in at least one of FR2 and / or FR4, and in particular at at least one of the Hallmark residues in FR2 and / or FR4 (again, including those at positions 108, 103 and / or 45).

[0320] As will be clear from the disclosure herein, it is also within the scope of the description to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs") of the Nanobodies of the description as defined herein, and in particular analogs of the Nanobodies of SEQ ID NO's 179 to 185 (see Table A-1). Thus, according to one aspect of the description, the term "Nanobody of the description" in its broadest sense also covers such analogs.

[0321] Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and / or added, compared to the Nanobodies of the description as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and / or in one or more of the CDR's. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and / or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

[0322] By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and / or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V HH domain (see Tables B-4 to B-7 for some non-limiting examples of such substitutions), although the description is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody of the description or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody of the description (i.e. to the extent that the Nanobody is no longer suited for its intended use) are included within the scope of the description. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies thus obtained.

[0323] For example, and depending on the host organism used to express the Nanobody or polypeptide of the description, such deletions and / or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).

[0324] As can be seen from the data on the V HH entropy and V HH variability given in Tables B-4 to B-7 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the description in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

[0325] The analogs are preferably such that they can bind to OX40L with an affinity (suitably measured and / or expressed as a K D -value (actual or apparent), a K A -value (actual or apparent), a k on -rate and / or a k off -rate, or alternatively as an IC 50 value, as further described herein) that is as defined herein for the Nanobodies of the description.

[0326] The analogs are preferably also such that they retain the favourable properties the Nanobodies, as described herein.

[0327] Also, according to one preferred aspect, the analogs have a degree of sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% or 99% or more; and / or preferably have at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2 or only 1 amino acid difference (as defined herein), with one of the Nanobodies of SEQ ID NOs: 179 to 185 (see Table A-1).

[0328] Also, the framework sequences and CDR's of the analogs are preferably such that they are in accordance with the preferred aspects defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and / or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and R at position 45; and / or (c) P, R or S at position 103.

[0329] One preferred class of analogs of the Nanobodies of the description comprise Nanobodies that have been humanized (i.e. compared to the sequence of a naturally occurring Nanobody of the description). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V HH with the amino acid residues that occur at the same position in a human V H domain, such as a human V H 3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and / or from a comparison between the sequence of a Nanobody and the sequence of a naturally occurring human V H domain.

[0330] The humanizing substitutions should be chosen such that the resulting humanized Nanobodies still retain the favourable properties of Nanobodies as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies thus obtained.

[0331] Generally, as a result of humanization, the Nanobodies of the description may become more "human-like", while still retaining the favorable properties of the Nanobodies of the description as described herein. As a result, such humanized Nanobodies may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V HH domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties pr...

Claims

1. An immunoglobulin single variable domain that can specifically bind to OX40L represented by SEQ ID NO: 175, in which: CDR1 is the amino acid sequence of SEQ ID NO: 135; and CDR2 is the amino acid sequence of SEQ ID NO: 149; and CDR3 is the amino acid sequence of SEQ ID NO: 163, wherein said immunoglobulin single variable domain has at least 80% amino acid identity to SEQ ID NO: 181, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the following amino acid residues: - at position 11: L, S, V, M, W, F, T, Q E, A, R, G, K, Y, N, P, I, preferably L, - at position 37: F, Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F or Y, - at position 44: E, Q, G, D, A, K, R, L, P, S, V, H, T, N, W, M, I, preferably G, E or Q, most preferably G - at position 45: L, R, P, H, F, G, Q, S, E, T, Y, C, I, D, V preferably L or R, - at position 47: F, L or W, G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D, preferably W , L or F, - at position 83: R, K, T, E, Q, N, S, I, V, G, M, L, A, D, Y, H, preferably K or R; most preferably K - at position 84: P, S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E, preferably P - at position 103: W, R, G, S, K, A, M, Y, L, F, T, N, V, Q, P, E, C, preferably W - at position 104: G, A, S, T, D, P, N, E, C, L, preferably G - at position 108: Q, L, R, P, E, K, S, T, M, A, H, preferably Q or L.

2. Immunoglobulin single variable domain according to claim 1, that essentially consists of a VHH sequence.

3. Immunoglobulin single variable domain according to any of claims 1 to 2, wherein said immunoglobulin single variable domain is chosen from the group consisting of - SEQ ID NO's: 200 to 205; - SEQ ID NO: 181.

4. Polypeptide that comprises or essentially consists of one or more immunoglobulin single variable domains according to any of claims 1 to 3, and optionally further comprises one or more other residues or binding units, optionally linked via one or more peptidic linkers.

5. Polypeptide according to claim 4, wherein said polypeptide is a bispecific polypeptide comprising or essentially consisting of one immunoglobulin single variable domain according to any of claims 1 to 3 and one other binding unit that is a VHH sequence, a humanized VHH sequence or partially humanized VHH sequence directed to an antigen that is different from OX40L, optionally linked via one or more peptidic linkers.

6. Polypeptide according to claim 4, in which said one or more other residues or binding units are chosen from the group consisting of immunoglobulin sequences, VHH sequences, humanized VHH sequences, or partially humanized VHH sequences.

7. Polypeptide according to claim 4 or 6, which is a multivalent construct, a multispecific construct, a multiparatopic construct, and / or has an increased half-life, compared to the corresponding immunoglobulin single variable domain according to any of claims 1 to 3 per se.

8. Polypeptide according to claim 7 in which said one or more other binding units provide the polypeptide with increased half-life, compared to the corresponding amino acid sequence according to any of claims 1 to 3 per se.

9. Polypeptide according to claim 8, in which said one or more other binding units that provide the polypeptide with increased half-life are chosen from the group consisting of binding units that can bind to serum albumin, human serum albumin, a serum immunoglobulin or IgG10. Nucleic acid or nucleotide sequence, that encodes an immunoglobulin single variable domain according to any of claims 1 to 3, or a polypeptide according to any of claims 4 to 9.

11. A host cell that expresses, or that under suitable circumstances is capable of expressing, an immunoglobulin single variable domain according to any of claims 1 to 3, or a polypeptide according to any of claims 4 to 9 and / or that comprises a nucleic acid or nucleotide sequence according to claim 10, wherein said host cell is a fungal cell, a prokaryotic cell, or ar eukaryotic cell line.

12. Composition comprising at least one immunoglobulin single variable domain according to any of claims 1 to 3, polypeptide according to any of claims 4 to 9, or nucleic acid or nucleotide sequence according to claim 10.

13. Method for producing an immunoglobulin single variable domain according to any of claims 1 to 3 or a polypeptide according to any of claims 4 to 9, - said method at least comprising the step of: expressing, in a suitable host cell or suitable in vitro expression system, a nucleic acid or nucleotide sequence according to claim 10, wherein said host cell is a fungal cell, a prokaryotic cell, or an eukaryotic cell line; optionally followed by isolating and / or purifying the immunoglobulin single variable domain according to any of claims 1 to 3, or the polypeptide according to any of claims 4 to 9; or - said method at least comprising the step of: cultivating and / or maintaining a host cell according to claim 11 under conditions that are such that said host cell expresses and / or produces at least one immunoglobulin single variable domain according to any of claims 1 to 3 or a polypeptide according to any of claims 4 to 9; optionally followed by isolating and / or purifying the immunoglobulin single variable domain according to any of claims 1 to 3 or said polypeptide according to any of claims 4 to 9.

14. Immunoglobulin single variable domain according to any of claims 1 to 3, a polypeptide according to any of claims 4 to 9, or a composition according to claim 12 for use as a medicament.

15. Immunoglobulin single variable domain according to any of claims 1 to 3, polypeptide according to any of claims 4 to 9 or composition according to claim 12 for the use in prevention and / or treatment of at least one inflammatory disease and / or disorder, asthma, allergic asthma, chronic colitis, Crohn's disease, inflammatory bowel disease, and / or arthrosclerosis.