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1. (WO2019025391) ANTIBODIES WITH FUNCTIONAL DOMAINS IN THE ELBOW REGION BETWEEN VARIABLE ADN CONSTANT DOMAIN
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CLAIMS

Antibody, or antigen binding fragment thereof, comprising a first polypeptide chain and a second polypeptide chain,

wherein said first polypeptide chain comprises in N- to C-terminal direction

(i) one or more variable domains;

(ii) a functional domain; and

(iii) one or more constant domains; and

wherein said second polypeptide chain comprises in N- to C-terminal direction

(iv) one or more variable domains forming antigen binding sites with the one or more variable domains of the first polypeptide chain;

(v) optionally, a functional domain; and

(vi) one or more constant domains,

characterized in that said functional domain (ii) of the first polypeptide chain does not comprise a fragment of the second polypeptide chain and said optional functional domain (v) of the second polypeptide chain does not comprise a fragment of the first polypeptide chain.

The antibody, or the antigen binding fragment thereof, according to claim 1 , wherein the second polypeptide chain comprises no functional domain (v) and wherein the C-terminus of the most C-terminal variable domain of the second polypeptide chain is directly coupled to the N-terminus of the most N-terminal constant domain of the second polypeptide chain.

Antibody, or antigen binding fragment thereof, comprising a first polypeptide chain and a second polypeptide chain,

wherein said first polypeptide chain comprises in N- to C-terminal direction

(i) one or more variable domains;

(ii) a functional domain and

(iii) one or more constant domains; and

wherein said second polypeptide chain comprises in N- to C-terminal direction

(iv) one or more variable domains forming antigen binding sites with the one or more variable domains of the first polypeptide chain; and

(v) one or more constant domains,

characterized in that the C-terminus of the most C-terminal variable domain of the second polypeptide chain is directly coupled to the N-terminus of the most N-terminal constant domain of the second polypeptide chain.

The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 3, wherein

(a) the one or more constant domains of the first polypeptide chain is/are heavy chain constant domains comprising at least a CH1 constant domain, the one or more variable domains of the first polypeptide chain is/are heavy chain variable domains VH, the constant domain of the second polypeptide chain is a light chain constant domain CL and the one or more variable domains of the second polypeptide chain is/are light chain variable domains VL; or

(b) the constant domains of the first polypeptide chain is a light chain constant domain CL, the one or more variable domains of the first polypeptide chain is/are light chain variable domains VL, the one or more constant domains of the second polypeptide chain is/are heavy chain constant domains comprising at least a CH1 constant domain and the one or more variable domains of the second polypeptide chain is/are heavy chain variable domains VH.

The antibody, or the antigen binding fragment thereof, according to claim 4, wherein the CH1 domain, and optionally any further heavy chain constant domain, is selected from the following classes: γ, α, μ, ε and δ; preferably γ, such as from lgG1 , lgC2, lgG3 or lgG4.

The antibody, or the antigen binding fragment thereof, according to claim 4 or 5, wherein the CL domain is selected from the following classes: κ and λ.

7. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 6, wherein the antibody, or the antigen binding fragment thereof, is a recombinant antibody or antigen binding fragment.

8. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain is not a linker, in particular not a GS-linker.

9. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 8, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of an Ig- like domain.

1 0. The antibody, or the antigen binding fragment thereof, according to claim 9, wherein the Ig-like domain is selected from the Ig-like domain(s) of PD1 , SLAM, CD2, CD3, CD4, CD8, CD1 9, CD22, CD33, CD80, or CD86, preferably CD4.

1 1 . The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 10, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a carrier domain, a reporter domain, a tag, a localization domain, an (independent) binding site, an enzyme or enzymatic domain, a receptor or a functional fragment thereof, or a ligand or a functional fragment thereof.

12. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a carrier domain.

1 3. The antibody, or the antigen binding fragment thereof, according to claim 12, wherein the carrier domain provides for conjugation of the antibody, or the antigen binding fragment thereof, to a drug or to an imaging agent.

Ί 4. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a reporter domain.

1 5. The antibody, or the antigen binding fragment thereof, according to claim 14, wherein the reporter domain comprises or consists of an amino acid sequence coding for GFP, YFP, RFP, CFP, luciferase, beta-galactosidase or peroxidase.

1 6. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a localization domain.

1 7. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of an enzyme or enzymatic domain, such as a catalytic domain.

18. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a tag.

1 9. The antibody, or the antigen binding fragment thereof, according to claim 18, wherein the tag is an affinity tag, a solubilization tag, a chromatography tag, an epitope tag or a fluorescence tag.

20. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a receptor or a functional fragment thereof, for example an Ig-like domain of a receptor.

21 . The antibody, or the antigen binding fragment thereof, according to claim 20, wherein the functional domain comprises or consists of (i) a receptor selected from the group consisting of PD1 , SLAM, LAIR1 , CTLA4, BTLA, TIM-3, TIGIT, CD200R1 , 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28, or (ii) a functional fragment of a receptor, such as an Ig-like domain of any one of the following receptors: PD1 , SLAM, LAIR1 , CTLA4, BTLA, TIM-3, TIGIT, CD200R1 , 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28.

22. The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of a ligand or a functional fragment thereof.

23. The antibody, or the antigen binding fragment thereof, according to claim 22, wherein the ligand is a cytokine and/or a ligand of PD1 , SLAM, LAIR1 , CTLA4, BTLA, TIM-3, TIGIT, CD200R1 , 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28.

24. The antibody, or the antigen binding fragment thereof, according to claim 22, wherein the ligand is selected from the group consisting of PD-L1 , PD-L2, Β7-Ί , B7-2, B7-H4 (B7 homolog), galectin-9, poliovirus receptor (PVR), OX-2 membrane glycoprotein, CD48, B7-H3 (B7 homolog), MHO, ICOS-L.

25. The antibody, or the antigen binding fragment thereof, according to claim 22, wherein the ligand is selected from the group consisting of the SIS family of cytokines, the SIG family of cytokines, the SCY family of cytokines, the Platelet factor-4 superfamily and intercrines, CC chemokine ligands (CCL)-I to -28, CXCL1 - CXCL1 7, XCL1 (lymphotactin-ct) and XCL2 (lymphotactin-β), fractalkine (or CX3CLI ); interferons, such as Type l lFN, Type II lFN, and Type III IFN, in particular IFN-a, lFN-β, IFN-γ, IFN-ε, IFN-K, IFN-ω, IL1 0R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12); interleukins, such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-1 3, IL-14, IL-15, IL-1 6, IL-1 7, IL-1 8, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL- 26, IL-27, IL-28, IL-29, IL-30, !L-31 , IL-32, IL-33, IL-34, IL-35, and IL-36; lymphokines, such as IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, and Interferon-gamma; tumor necrosis

factors, such as CD40LG (TNFSF5); CD70 (TNFSF7); EDA; FASLG (TNFSF6); LTA (TNFSF1 ); LTB (TNFSF3); TNF, TNFa, TNFSF4 (OX40L); TNFSF8 (CD1 53); TNFSF9; TNFSF10 (TRAIL); TNFSF1 1 (RANKL); TNFSF12 (TWEAK); TNFSF13; TNFSF13B; TNFSF14; TNFSF1 5; and TNFSF18; and colony stimulating factors, such as CSF1 (macrophage colony-stimulating factor), CSF2 (GM-CSF), CSF3 (G-CSF), as well as synthetic CSFs, such as Promegapoietin.

The antibody, or the antigen binding fragment thereof, according to claim 1 1 , wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain comprises or consists of an (independent) binding site.

The antibody, or the antigen binding fragment thereof, according to claim 26, wherein the (independent) binding site comprises or consists of a receptor or a functional fragment thereof comprising a binding site, a ligand or a functional fragment thereof comprising a binding site, a CD molecule or a functional fragment thereof, a single chain antibody or an antigen binding fragment thereof, an antigen or a functional fragment thereof, or a tag comprising a binding site.

The antibody, or the antigen binding fragment thereof, according to claim 27, wherein the (independent) binding site comprises or consists of a single chain antibody (such as scFv or VHH) or an antigen binding fragment thereof, or an antigen or a functional fragment thereof.

The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 28, wherein the functional domain comprises or consists of an Ig-like domain, an scFv, a VHH or a Strep-tag.

The antibody, or the antigen binding fragment thereof, according to claim 29, wherein
the functional domain comprises or consists of an amino acid sequence as set forth in any of SEQ ID NOs 1 3 - 20, or a functional sequence variant thereof having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity.

31 . The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 30, wherein the fi rst polypeptide chain comprises one single variable domai n (i) N- terminal of the functional domain (ii) and the second polypeptide chain comprises one single variable domai n (iv) N-terminal of the optional functional domain (v) or N- termi nal of the one or more constant domains (vi), the one single variable domain (iv) of the second polypeptide chain forming an antigen bi nding site with the variable domain (i) of the first polypeptide chai n.

32. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 30, wherein the first polypeptide chain comprises two or more variable domains (i) N-terminal of the functional domain (ii) and the second polypeptide chain comprises two or more variable domains (iv) N-terminal of the optional functional domain (v) or N-terminal of the one or more constant domains (vi), the two or more variable domains (iv) of the second polypeptide chain forming antigen binding sites with the two or more variable domains (i) of the first polypeptide chain.

33. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 32, wherein the first polypeptide chain comprises a si ngle chain antibody, such as an scFv or a single domain antibody, for example a VHH, N-terminal of the most N- terminal variable domain (i) and/or C-terminal of the most C-terminal constant domain (ii i).

34. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 33, wherein the second polypeptide chain comprises a single chain antibody, such as an scFv or a single domain antibody, for example a VHH, N-terminal of the most N- terminal variable domain (i) and/or C-terminal of the most C-terminal constant domain (vi).

35. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 34, wherein the first polypeptide chain and the second polypeptide chain each comprise one single constant domain, in particular a CL domain and a CH1 domain, respectively.

36. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 35, wherein the first polypeptide chain or the second polypeptide chain comprises one single constant domain, in particular a CL domain; and the other of the first polypeptide chain or the second polypeptide chain comprises a CH I domain and one or more further constant domains, such as a CH2 and/or CH3 domain.

37. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 36, wherein the antibody , or the antigen binding fragment thereof, comprises an Fc moiety or an Fc region.

38. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 37, wherein the first polypeptide chain and/or the second polypeptide chain comprises one or more linkers.

39. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 38, wherein the first polypeptide chain and/or the second polypeptide chain does not comprise any linkers.

40. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 39, wherein the first polypeptide chain comprises or consists of V-D-CH1 , wherein V is a variable domain (i);

D is a functional domain (ii); and

CH1 is a CH1 constant domain (iii).

41 . The antibody, or the antigen binding fragment thereof, according to claim 40, wherein the first polypeptide chain comprises or consists of V-D-CH1 -CH2-CH3, wherein CH2 and CH3 are a CH2 constant domain and a CH3 constant domain, respectively.

42. The antibody, or the antigen binding fragment thereof, according to any one of claims 39 - 41 , wherein the first polypeptide chain comprises or consists of (V)A-D-CH 1 with A being an integer from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3 and even more preferably 1 or 2 and wherein the variable domains V may be coupled to each other directly or via a linker.

43. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 41 , wherein the second polypeptide chain comprises or consists of V-CL, wherein V is a variable domain (iv); and

CL is a constant domain (vi).

44. The antibody, or the antigen binding fragment thereof, according to claim 43, wherein the second polypeptide chain comprises or consists of (V)A-CL with A being an integer from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3 and even more preferably 1 or 2 and wherein the variable domains V may be coupled to each other directly or via a linker.

45. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 44, wherein the variable domain (i) of the first polypeptide chain comprises or consists of an amino acid sequence as set forth in any one of SEQ I D NOs 1 , 5, 8 or 1 0, or a functional sequence variant thereof having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity.

46. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 45, wherein the variable domain (iv) of the second polypeptide chain comprises or consists of an amino acid sequence as set forth in any one of SEQ I D NOs 2, 6, 9 or 1 1 , or a functional sequence variant thereof having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity.

47. The antibody, or the antigen bindi ng fragment thereof, according to any one of claims 1 - 46, wherein the antibody, or the antigen binding fragment thereof, comprises one single or two first polypeptide chains and one single or two second polypeptide chai ns.

48. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 47, wherein the first polypeptide chain and the second polypeptide chain are linked by a disulfide bond, thereby formi ng a pair.

49. The antibody, or the antigen binding fragment thereof, according to claim 47 or 48, wherein the antibody, or the antigen binding fragment thereof, comprises two first polypeptide chains and two second polypeptide chains and wherei n the two first polypeptide chains and/or the two second polypeptide chains are linked by one or more, such as two, disulfide bonds.

50. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 49, wherein the antibody, or the antigen binding fragment thereof, is bivalent for each specificity/antigen.

51 . The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 49, wherein the antibody, or the antigen binding fragment thereof, is monovalent for each specificity (antigen).

52. The antibody, or the antigen binding fragment thereof, according to claim 50 or 51 , wherein the antigen binding site(s) formed by the one or more variable domains of the first pair of first and second polypeptide chains and the antigen binding site(s) formed by the one or more variable domains of the second pair of first and second polypeptide chains are the same or disti nct.

53. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 52, wherein the antibody, or the antigen binding fragment thereof, comprises at least two functional domains, which may be the same or distinct.

54. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 53, wherein the antibody, or the antigen binding fragment thereof, comprises two first polypeptide chains and two second polypeptide chains forming a first and a second pair of first and second polypeptide chains, and wherein the first pair of first and second polypeptide chains comprises at least one functional domain(s) and/or the second pair of first and second polypeptide chains comprises at least one functional domain(s).

55. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 54, wherein the antibody, or the antigen binding fragment thereof, is derived from an IgG-like antibody, a Fab or a F(ab)2.

56. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 55, wherein the two or more variable domains (i) and (iv) of the antibody, or the antigen binding fragment thereof, are derived from a monoclonal antibody.

57. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 56, wherein the variable domains and/or the constant domains of the antibody, or the antigen binding fragment thereof, are human or humanized.

58. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 57, wherein the (additional) functional domain(s) of the antibody, or the antigen binding fragment thereof, comprise(s) or consist(s) of an amino acid sequence, which is human or humanized.

59. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 58, wherein the first polypeptide chain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs 53, 55, 56, 58, 59, 60, 61 , 62, 64, 65, 66, 68, 69 or 70, or a functional sequence variant thereof having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity, and/or the second polypeptide chain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs 54, 57, 63, or 67, or a functional sequence variant thereof having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity.

The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 59, wherei n the first and/or the second polypeptide chain does not comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs 75 - 92 or 96 - 1 12, preferably, the first and/or the second polypeptide chain does not comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs 75 - 95, more preferably, the functional domain of the first and/or the second polypeptide chain does not comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs 1 1 3 - 1 30, even more preferably, the functional domain of the first and/or the second polypeptide chain does not comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs 1 1 3 - 1 33, and most more preferably, the first and/or the second polypeptide chain does not comprise or consist of an amino acid sequence as set forth i n any one of SEQ ID NOs 75 - 1 33.

Nucleic acid molecule comprising a first polynucleotide encoding a first polypeptide chain of the antibody, or antigen-binding fragment thereof, according to any one of claims 1 - 60 and/or a second polypeptide encoding the second polypeptide chain of the antibody, or antigen-binding fragment thereof, according to any one of claims 1 - 60.

Nucleic acid molecule comprisi ng a polynucleotide encodi ng the first polypeptide chain and the second polypeptide chain of the antibody, or antigen-binding fragment thereof, according to any one of claims 1 - 60.

The nucleic acid molecule according to claim 61 or 62, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

64. Vector comprising the nucleic acid molecule according to any one of claims 61

65. Host cell comprising the nucleic acid molecule according to any one of claims 61 - 63 or the vector according to claim 64.

66. Composition comprising the antibody, or antigen-binding fragment thereof, according to any one of claims 1 - 60, the nucleic acid molecule according to any one of claims

61 - 63, the vector according to claim 64 or the host cell according to claim 65.

67. A process for produci ng the antibody, or antigen-binding fragment thereof, according to any one of claims 1 - 60 comprising:

a) transforming an eukaryotic host cell by incorporating one or more nucleic acid molecule(s) according any one of claims 61 - 63 encoding a first polypeptide chain and a second polypeptide chain;

b) cultivating the host cell under suitable conditions so that said nucleic acid molecules are expressed;

c) causing or allowing said first and second polypeptide chains to combine to form the antibody, or antigen-binding fragment thereof; and

d) optionally, purifying the antibody, or antigen-binding fragment thereof, from the culture medium.

68. The antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 60, the nucleic acid molecule according to any one of claims 61 - 63, the vector accordi ng to claim 64, the host cell according to claim 65, or the composition according to claim 66 for use in medicine.

69. Method of preventing or treating a disease or disorder in a subject comprising the step of administering the antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 60, the nucleic acid molecule accordi ng to any one of claims 61 - 63, the vector according to claim 64, the host cell according to claim 65, or the composition according to claim 66 to the subject.

70. An assay for detecting an antigen or for quantification of antigen bi nding comprising: incubating an antigen with the antibody, or the antigen binding fragment thereof, according to any one of claims 1 - 60 under conditions which permit bindi ng of the antigen to the polyvalent antibody; and

detecti ng antigen-antibody bi nding.