ANTIBODIES WITH FUNCTIONAL DOMAINS IN THE ELBOW REGION BETWEEN VARIABLE AND CONSTANT DOMAIN

Abstract

The present invention provides engineered antibodies and antigen binding fragments, in which an additional functional domain is inserted into the elbow region of the antibody or antigen binding fragment. The present invention also provides nucleic acid molecules, such as vectors, encoding such antibodies and antigen binding fragments, host cells and compositions comprising such antibodies, antigen binding fragments or nucleic acid molecules and uses thereof. For example a multispecific antibody format is provided, in which an additional binding site (specificity) is inserted into the elbow region of an antibody or antigen binding fragment.

Claims

1. 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.

2. 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.

3. 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.

4. The antibody, or the antigen binding fragment thereof, according to claim 1, 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.

5. 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: , , , and ; preferably , such as from IgG1, IgG2, IgG3 or IgG4.

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

7. The antibody, or the antigen binding fragment thereof, according to claim 1, wherein: (a) the antibody, or the antigen binding fragment thereof, is a recombinant antibody or antigen binding fragment; (b) 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; or (c) 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.

8.-10. (canceled)

11. The antibody, or the antigen binding fragment thereof, according to claim 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, 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 11, wherein: (a) 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; (b) 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; (e) 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; (d) 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; (e) 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; (f) 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; (g) 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; or (h) 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.

13.-28. (canceled)

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

30. 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 13-20, 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 claim 1, wherein (a) the first polypeptide chain comprises one single variable domain (i) N-terminal of the functional domain (ii) and the second polypeptide chain comprises one single variable domain (iv) N-terminal of the optional functional domain (v) or N-terminal of the one or more constant domains (vi), the one single variable domain (iv) of the second polypeptide chain forming an antigen binding site with the variable domain (i) of the first polypeptide chain; or (b) 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.

32. (canceled)

33. The antibody, or the antigen binding fragment thereof, according to claim 1, wherein; (a) the first 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 (iii); (b) 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); (c) 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; or (d) 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 CH1 domain and one or more further constant domains, such as a CH2 and/or CH3 domain.

34.-36. (canceled)

37. The antibody, or the antigen binding fragment thereof, according to claim 1, 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 claim 1, 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 claim 1, 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 claim 1, 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. (canceled)

42. The antibody, or the antigen binding fragment thereof, according to claim 39, wherein the first polypeptide chain comprises or consists of (V).sub.A-D-CH1 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 claim 1, 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).sub.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 claim 1, 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 ID NOs 1, 5, 8 or 10, 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 claim 1, 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 ID NOs 2, 6, 9 or 11, 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 binding fragment thereof, according to claim 1, wherein the antibody, or the antigen binding fragment thereof, comprises one single or two first polypeptide chains and one single or two second polypeptide chains.

48.-49. (canceled)

50. The antibody, or the antigen binding fragment thereof, according to claim 1, wherein; (a) the antibody, or the antigen binding fragment thereof, is bivalent for each specificity/antigen; or (b) the antibody, or the antigen binding fragment thereof, is monovalent for each specificity (antigen).

51.-52. (canceled)

53. The antibody, or the antigen binding fragment thereof, according to claim 1, wherein; (a) the antibody, or the antigen binding fragment thereof, comprises at least two functional domains, which may be the same or distinct; (b) 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); (c) the antibody, or the antigen binding fragment thereof, is derived from an IgG-like antibody, a Fab or a F(ab).sub.2; (d) the two or more variable domains (i) and (iv) of the antibody, or the antigen binding fragment thereof, are derived from a monoclonal antibody; (e) the variable domains and/or the constant domains of the antibody, or the antigen binding fragment thereof, are human or humanized; (f) 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; (g) 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; or (h) 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 96-112, 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 113-130, 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 113-133, 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 in any one of SEQ ID NOs 75-133.

54.-60. (canceled)

61. A nucleic acid molecule comprising a first polynucleotide encoding a first polypeptide chain of the antibody, or antigen-binding fragment thereof, according to claim 1 and/or a second polypeptide encoding the second polypeptide chain of the antibody, or antigen-binding fragment thereof, according to claim 1.

62.-63. (canceled)

64. A vector comprising the nucleic acid molecule according to claim 61.

65. A host cell comprising the nucleic acid molecule according to claim 61.

66. A composition comprising the antibody, or antigen-binding fragment thereof, according to claim 1, and a pharmaceutically acceptable carrier, excipient, or diluent.

67. A process for producing the antibody, or antigen-binding fragment thereof, according to claim 1 comprising: a) transforming an eukaryotic host cell by incorporating one or more nucleic acid molecule(s) encoding the first polypeptide chain and the second polypeptide chain; b) cultivating the host cell under suitable conditions so that said one or more nucleic acid molecule(s) is/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. (canceled)

69. A 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, or a nucleic acid encoding the same, according to claim 1 to the subject.

70. An assay for detecting an antigen or for quantification of antigen binding comprising: a) incubating an antigen with the antibody, or the antigen binding fragment thereof, according to claim 1 under conditions which permit binding of the antigen to the antibody or the antigen binding fragment thereof; and b) detecting antigen-antibody binding.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0284] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0285] FIG. 1 (A) classical monospecific antibody comprising two heavy chains (grey), each with a single variable domain VH and three constant domains CH1, CH2 and CH3, and two light chains (white), each with a single variable domain VL and a single constant domain CL. The elbow region is indicated by arrows. [0286] (B) Preferred examples of antibodies according to the present invention (n-elbow-insert Ig molecules; IEI Ig) derived from the classical monospecific antibody shown in (A): antibody comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; antibody comprising one single (additional) functional domain in the elbow region of each light chain but no elbow insert in the heavy chain; antibody comprising one single (additional) functional domain in the elbow region of each heavy chain and of each light chain; antibody comprising two (additional) functional domains in the elbow region of each heavy chain but no elbow insert in the light chain; and antibody comprising two (additional) functional domains in the elbow region of each light chain but no elbow insert in the heavy chain. [0287] (C) Preferred examples of antigen binding fragments and antibodies according to the present invention (In-elbow-insert Ig molecules; IEI Ig) derived from antibody fragments or from bispecific antibodies: F(ab) fragment comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; F(ab).sub.2 fragment comprising one single (additional) functional domain in the elbow region of each light chain but no elbow insert in the heavy chain; Fab fragment comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; Fab fragment comprising one single (additional) functional domain in the elbow region of each light chain but no elbow insert in the heavy chain; CrossMab/Knobs-in-holes/orthogonal Fab/Fab-arm exchange antibodies comprising one single (additional) functional domain in the elbow region of one single heavy chain but no elbow insert in the light chains and in the other heavy chain; scFv-(H)gG comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; IgG(H)-scFv comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; scFv-(L)IgG comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; IgG(L)-scFv comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain; and DVD-Ig comprising one single (additional) functional domain in the elbow region of each heavy chain but no elbow insert in the light chain.

[0288] FIG. 2 shows for Example 1 a scheme of the seven antibody constructs according to the present invention (C2 to C8) compared to scaffold antibodies (GCE536, C1).

[0289] FIG. 3 shows for Example 2 the EC50 values determined by non-linear regression analysis of binding values (OD) and relative antibody concentrations in the ELISA tests made using a set of antigens or anti-domain antibodies. The constructs were also tested for binding to IEs (isolate 9215) and binding values (%) at a concentration of 1 g/ml are shown.

[0290] FIG. 4 shows for Example 3 the SPR binding curves of C4 and different controls to GM-CSF and collagen. C4 and C5 that use GCE536 as scaffold bind to GM-CSF, but only C4 is then bound by collagen. Non-specific FI174 and collagen-specific MGD.sup.UCA antibodies don't show any specific binding signal.

[0291] FIG. 5 shows for Example 3 the SPR binding curves of C4 and different controls to GM-CSF and collagen. C4 and GCE536 bind to GM-CSF, but only C4 is then recognized by collagen. Collagen-specific MGD.sup.UCA antibody binds to collagen only. Control antibody TT107 is a T-specific monoclonal antibody, which does not show any specific binding signal in SPR experiment.

[0292] FIG. 6 shows for Example 4 the ELISA binding curves of C5, C5b, C6 and C6b constructs to GM-CSF or by anti-PD1 or anti-SLAM antibodies, compared to the scaffold antibody (GCE536). PD1-containing C5 and C5b and SLAM-containing C6 and C6b constructs are recognized by anti-PD1 or anti-SLAM antibodies, respectively. All the constructs bind to GM-CSF as GCE536. The presence of linkers in C5b and C6 does not affect binding.

[0293] FIG. 7 shows for Example 6 the ELISA binding curve of a Strep-tactin antibody to C9 construct compared to the scaffold C1b. The Twin Strep-tag inserted in the elbow of C9 construct is specifically recognized by the Strep-tactin antibody.

[0294] FIG. 8 shows for Example 7 a scheme of the four additional antibody constructs (C9 to C12) compared to the scaffold antibody (F174).

[0295] FIG. 9 shows for Example 8 the SPR binding curves of C9 and C10 and different controls to H1 and TT. C9 and C10 show dual binding to both H1 and TT. TT107 is a TT-specific monoclonal antibody.

[0296] FIG. 10 shows for Example 8 the SPR binding curves of C11 and C12 and different controls to H1 and RSV F protein. C11 and C12 show dual binding to both H1 and RSV F protein. MPE8 is a RSV F protein-specific monoclonal antibody.

EXAMPLES

[0297] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Design and Construction of Antibody Variants Inserting Different Ig-Like Functional Domains into the Elbow Region of Scaffold Antibodies

[0298] To investigate the effect of distinct (additional) functional domains inserted into the elbow region of an antibody on antibody specificity, seven different constructs (named C2-C8) were designed, in which unmutated LAIR1 (SEQ ID NO: 12), mutated LAIR1 (SEQ ID NO: 13) or other Ig-like domains were inserted into the elbow region of an antibody that was used as a scaffold. Constructs C2-C3 have the same complete constant region of the heavy chain as construct C1 (VH: SEQ ID NO: 5, VL: SEQ ID NO: 6, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 7). Constructs C4-C6 have the same complete constant region of the heavy chain as the antibody GCE536 (VH: SEQ ID NO: 1, VL: SEQ ID NO: 2, heavy chain constant region: SEQ ID NO: 3, light kappa chain constant region: SEQ ID NO: 4; Piccoli, L., et al. Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis. Nature communications 6, 7375 (2015)). Constructs C7-C8 have the same complete constant region of the heavy chain as construct C1b (VH: SEQ ID NO: 8, VL: SEQ ID NO: 9, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4). The light chain of the constructs was not modified in comparison to the scaffold antibodies. All construct were finally expressed as monoclonal antibodies (heavy and light chains).

[0299] The following constructs were produced and are shown schematically in FIG. 2: [0300] 1. C1 (VH: SEQ ID NO: 5, VL: SEQ ID NO: 6, heavy chain constant region: SEQ ID NO: 3, light chain lambda constant region: SEQ ID NO: 7) is a recombinant monospecific antibody for control purposes. C1 is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain (VH3-30); the expression product of a D (diversity) gene segment of a heavy chain variable domain (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain (JH6); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain and the expression product of a J (Joining) gene segment element of a light chain variable domain; the expression product of a C (constant) gene segment of a light chain lambda constant region. [0301] 2. C1b (VH: SEQ ID NO: 8, VL: SEQ ID NO: 9, heavy chain constant region: SEQ ID NO: 3, light chain kappa constant region: SEQ ID NO: 4) is a recombinant monospecific antibody for control purposes. C1 is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain (VH3-20); the expression product of a D (diversity) gene segment of a heavy chain variable domain (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain (H3; the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain and the expression product of a J (Joining) gene segment element of a light chain variable domain; the expression product of a C (constant) gene segment of a light chain kappa constant region. [0302] 3. In construct C2 a mutated LAIR-1 fragment (LAIR1.sup.mut; SEQ ID NO: 13) was inserted into the elbow region of recombinant monospecific antibody C1 (see above). In the mutated LAIR1 fragment, the binding of LAIR1 to collagen is abolished, but the mutated LAIR1 fragment binds strongly to P. falciparum infected erythrocytes (Tan, J., Pieper, K., Piccoli, L., et al. A LAIR insertion generates broadly reactive antibodies against malaria variant antigens. Nature 529, 105-109 (2016); WO 2016/207402 A1). Construct C2 (complete heavy chain: SEQ ID NO: 53, complete light chain: SEQ ID NO: 54) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of C1 (VH3-30); the expression product of a D (diversity) gene segment of a heavy chain variable domain of C1 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of C (JH6); the expression product of the mutated LAIR-1 fragment of MGD21 (LAIR.sup.D21); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of C1 and the expression product of a j (Joining) gene segment element of a light chain variable domain of C1; the expression product of a C (constant) gene segment of a light chain constant region. [0303] 4. In construct C3 an unmutated LAIR-1 fragment (LAIR1.sup.gen; SEQ ID NO: 12) was inserted into the elbow region of recombinant monospecific antibody C1 (see above). Construct C3 (complete heavy chain: SEQ ID NO: 55, complete light chain: SEQ ID NO: 54) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of C (VH3-30); the expression product of a D (diversity) gene segment of a heavy chain variable domain of C1 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of C1 (JH6); the expression product of the unmutated LAIR-1 fragment (LAIR1.sup.gen); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of C1 and the expression product of a J (Joining) gene segment element of a light chain variable domain of C1; the expression product of a C (constant) gene segment of a light chain constant region. [0304] 5. In construct C4 an unmutated LAIR-1 fragment (LAIR1.sup.gen; SEQ ID NO: 13) was inserted into the elbow region of antibody GCE536. Construct C4 (complete heavy chain: SEQ ID NO: 56, complete light chain: SEQ ID NO: 57) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of GCE536 (VH1-46); the expression product of a D (diversity) gene segment of a heavy chain variable domain of GCE536 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of GCE536 (JH6); the expression product of the unmutated LAIR-1 fragment (LAIR1.sup.gen); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of GCE536 and the expression product of a J (Joining) gene segment element of a light chain variable domain of GCE536; the expression product of a C (constant) gene segment of a light chain constant region. [0305] 6. In construct C an extracellular Ig-like domain of PD1 molecule (PD1; SEQ ID NO: 14) was inserted into the elbow region of antibody GCE536. Construct C5 (complete heavy chain: SEQ ID NO: 58, complete light chain: SEQ ID NO: 57) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of GCE536 (VH1-46); the expression product of a D (diversity) gene segment of a heavy chain variable domain of GCE536 (D); the expression product of a J Coining) gene segment element of a heavy chain variable domain of GCE536 (JH6); the expression product of an extracellular Ig-like domain of PD1 molecule (PD1); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of GCE536 and the expression product of a J (Joining) gene segment element of a light chain variable domain of GCE536; the expression product of a C (constant) gene segment of a light chain constant region. [0306] 7. In construct C5b an extracellular Ig-like domain of PD1 molecule (PD1; SEQ ID NO: 14) with flanking intronic sequences (15-mer JH-PD1; SEQ ID NO: 49 and 15-mer PD1-CH1 linkers; SEQ ID NO: 50) was inserted into the elbow region of antibody GCE536. Construct C5 (complete heavy chain: SEQ ID NO: 59, complete light chain: SEQ ID NO: 57) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of GCE536 (VH1-46); the expression product of a D (diversity) gene segment of a heavy chain variable domain of GCE536 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of GCE536 (JH6); the expression product of a linker (15-mer JH-PD1); the expression product of an extracellular Ig-like domain of PD1 molecule (PD1); the expression product of a linker (15-mer PD1-CH); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of GCE536 and the expression product of a J (Joining) gene segment element of a light chain variable domain of GCE536; the expression product of a C (constant) gene segment of a light chain kappa constant region. [0307] 8. In construct C6 an extracellular Ig-like domain of SLAM molecule (SLAM; SEQ ID NO: 15) was inserted into the elbow region of antibody GCE536. Construct C6 (complete heavy chain: SEQ ID NO: 60, complete light chain: SEQ ID NO: 57) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of GCE536 (VH1-46); the expression product of a D (diversity) gene segment of a heavy chain variable domain of GCE536 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of GCE536 (JH6); the expression product of an extracellular Ig-like domain of SLAM molecule (SLAM); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of GCE536 and the expression product of a J (Joining) gene segment element of a light chain variable domain of GCE536; the expression product of a C (constant) gene segment of a light chain constant region. [0308] 9. In construct C6b an extracellular Ig-like domain of SLAM molecule (SLAM; SEQ ID NO: 15) with flanking intronic sequences (15-mer JH-SLAM; SEQ ID NO: 51 and 15-mer SLAM-CH1 linkers; SEQ ID NO: 52) was inserted into the elbow region of antibody GCE536. Construct C6 (complete heavy chain: SEQ ID NO: 61, complete light chain: SEQ ID NO: 57) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of GCE536 (VH1-46); the expression product of a D (diversity) gene segment of a heavy chain variable domain of GCE536 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of GCE536 (JH6); the expression product of a linker (15-mer JH-SLAM); the expression product of an extracellular Ig-like domain of SLAM molecule (SLAM); the expression product of a linker (15-mer SLAM-CH); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of GCE536 and the expression product of a J (Joining) gene segment element of a light chain variable domain of GCE536; the expression product of a C (constant) gene segment of a light chain kappa constant region. [0309] 10. In construct C7 an extracellular Ig-like domain of PD1 molecule (PD1; SEQ ID NO: 14) was inserted into the elbow region of recombinant monospecific antibody C1b (see above). Construct C7 (complete heavy chain: SEQ ID NO: 62, complete light chain: SEQ ID NO: 63) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain (VH3-20); the expression product of a D (diversity) gene segment of a heavy chain variable domain (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain (H3); the expression product of an extracellular Ig-like domain of PD1 molecule (PD1); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain and the expression product of a J (Joining) gene segment element of a light chain variable domain; the expression product of a C (constant) gene segment of a light chain constant region. [0310] 11. In construct C8 an extracellular Ig-like domain of SLAM molecule (SLAM; SEQ ID NO: 15) was inserted into the elbow region of recombinant monospecific antibody C1b (see above). Construct C8 (complete heavy chain: SEQ ID NO: 64, complete light chain: SEQ ID NO: 63) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain (VH3-20); the expression product of a D (diversity) gene segment of a heavy chain variable domain (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain (JH3); the expression product of an extracellular Ig-like domain of SLAM molecule (SLAM); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain and the expression product of a J (Joining) gene segment element of a light chain variable domain; the expression product of a C (constant) gene segment of a light chain constant region.

Example 2: Ig-Like Domains can be Inserted in the Elbow Region of Antibodies Resulting in Functional Antibodies

[0311] The eight antibody constructs described in Example 1 were produced recombinantly by transient transfection. To this end, antibody heavy and light chains were cloned into human IgG1, Ig and Ig expression vectors and expressed by transient transfection of Expi293F Cells (ThermoFisher Scientific) using polyethylenimine (PEI). Cell lines were routinely tested for mycoplasma contamination.

[0312] Next, the antibody constructs C1-C8 and control antibody GCE536 (see Example 1) were tested for staining of 9215 IEs (infected erythrocytes) and binding values (%) at 1 g/ml antibody concentration were calculated by interpolation of binding curves fitted to a sigmoidal curve model (Graphpad Prism 6). In addition, binding to recombinant human collagen, anti-human LAIR1 antibody, recombinant human GM-CSF, an anti-PD1 and an anti-SLAM antibody was tested by ELISA. Briefly, total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody constructs, ELISA plates were coated with 2 g ml.sup.1 of type I recombinant human collagen (Millipore, CC050), 2 g ml.sup.1 of an anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 g ml.sup.1 of recombinant human GM-CSF (Gentaur), 2 g ml.sup.1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems, AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fc fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm.

[0313] Results of the different binding studies are shown in FIG. 3. Antibody constructs carrying LAIR1 stained IEs and were recognized by an anti-LAIR1 antibody. Insertion of LAIR1, PD1 or SLAM Ig-like domains into the elbow region of an anti-GM-CSF antibody did not affect binding to GM-CSF (constructs C4-C6), indicating that this site is permissive for insertions of different domains without affecting the original antibody specificity. In contrast, insertion of LAIR1 into the CDR3 abolished binding to GM-CSF (data not shown). Accordingly, the elbow region is permissive for insertions of different domains, without affecting the original antibody specificity.

Example 3: Constructs Containing an Ig-Like Domain in the Elbow can Simultaneously Bind to Two Different Antigens

[0314] To test whether the bispecific constructs carrying a binding site in the elbow region, are able to simultaneously bind with both specificities, simultaneous binding was investigated by surface plasmon resonance (SPR). To this end, bispecific construct C4, which has a V(D) region specific for GM-CSF and carries unmutated LAIR1 (binding site for collagen) in the elbow, was tested for simultaneous binding to GM-CSF and collagen by surface plasmon resonance (SPR).

[0315] In one experiment, GM-CSF was immobilized on the surface of a sensor chip and the constructs were injected, followed by injection of collagen. Antibodies C5 and GCE536 (cf. above), antibody MGD.sup.UCA (contains unmutated LAIR1 and is, thus, able to bind collagen; Tan, J., Pieper, K., Piccoli, L., et al. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature 529, 105-109 (2016); VH: SEQ ID NO: 71, VL: SEQ ID NO: 72, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) and antibody F1174 (specific for influenza H1 hemagglutinin; Pappas, L., et al. Rapid development of broadly influenza neutralizing antibodies through redundant mutations. Nature 516, 418-422 (2014); (VH: SEQ ID NO: 10, VL: SEQ ID NO: 11, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) were used as controls. Briefly, GM-CSF (200 nM) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized onto an ethyl(dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) pre-activatedProteOnsensor chip (Bio-Rad) through amine coupling; unreacted groups were blocked by injection of 1M ethanolamine HCL. HEPES buffered saline (HBS) (10 mMHEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20) was used as a running buffer. All injections were made at a flowrate of 100 l/min. Monoclonal antibodies were diluted in HBS to 10 nM and injected for 240 s onto the GM-CSF-coated chip, followed by injection of collagen (50 nM) for 120 s. One channel of the chip was injected with HBS and used as reference for the analysis. Each binding interaction of the monoclonal antibodies to GM-CSF and collagen was assessed using a ProteONXPR36 instrument (Bio-Rad) and the data were processed with ProteOn Manager software.

[0316] Results are shown in FIG. 4. Both constructs C4 and C5, which use GCE536 as scaffold (see Example 1), bind to GM-CSF. Of C4 and C5, only C4 (which carries LAIR1 in the elbow regionwhereas C5 carries an Ig-like domain of PD1 in the elbow region) is then bound by collagen. Monospecific anti-GM-CSF antibody GCE536 binds only to GM-CSF, but not to collagen. Anti-H1 antibody FI74 and collagen-specific MGD.sup.UCA antibody do not show any specific binding signal. In summary, those data show that bispecific construct C4 bound to GM-CSF and then collagen bound to the LAIR1 domain of C4.

[0317] In a second experiment, protein A was used to capture the construct followed by co-injection of the analytes (GM-CSF followed by collagen). In this experiment antibodies GCE536 and MGD.sub.UCA (all described in Examples 1 and 2) and antibody TT107 (specific for tetanus toxoid (TT); VH: SEQ ID NO: 73, VL: SEQ ID NO: 74, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) were used as controls. Briefly, Protein A (25 g/ml) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized onto an ethyl(dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) pre-activatedProteOnsensor chip (Bio-Rad) through amine coupling; unreacted groups were blocked by injection of 1M ethanolamine HCl. HEPES buffered saline (HBS) (10 mMHEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20) was used as a running buffer. All injections were made at a flowrate of 100 l/min. Monoclonal antibodies were diluted in HBS to 10 nM and injected for 240 s onto the Protein A-coated chip for capturing, followed by co-injection of GM-CSF collagen (50 nM) immediately followed by collagen (50 nM) for total 110 s. One channel of the chip was injected with HBS and used as reference for the analysis. Each binding interaction of the monoclonal antibodies to GM-CSF and collagen was assessed using a ProteONXPR36 instrument (Bio-Rad) and the data were processed with ProteOn Manager software.

[0318] Results are shown in FIG. 5. Construct C4 and GCE536 bind to GM-CSF. However, only C4 is then recognized by collagen. Collagen-specific MGD.sup.UCA antibody binds to collagen only. Control antibody TT107 is a TT (tetanus toxoid)-specific monoclonal antibody, which does not show any specific binding signal in this SPR experiment. In summary those results show that constructs containing a (additional) functional domain, such as an Ig-like domain, in the elbow region can simultaneously bind to (i) the binding partner of the elbow binding site and (ii) the antigen recognized by the variable domains of the scaffold antibody.

Example 4: Ig-Like Domains can be Inserted Together with Flanking Linkers in the Elbow Region of Antibodies Resulting in Functional Antibodies

[0319] The antibody constructs C5b and C6b contain the same domain (either PD1 or SLAM) inserted in the elbow in C5 and C6 constructs, respectively, with two additional 15-mer amino acid linkers inserted between JH and the domain, and between the domain and CH1. The constructs were tested for binding to recombinant human GM-CSF, an anti-PD1 and an anti-SLAM antibody by ELISA. Briefly, total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody constructs, ELISA plates were coated with 1 g ml.sup.1 of recombinant human GM-CSF (Gentaur), 2 g ml.sup.1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems, AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fc fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm.

[0320] Results of the different binding studies are shown in FIG. 6. Insertion of PD1 or SLAM Ig-like domains with flanking linkers into the elbow region of an anti-GM-CSF antibody did not affect binding to GM-CSF, indicating that this site is permissive for insertions also of different domains with flanking linkers without affecting the original antibody specificity. In addition, the presence of the linkers did not alter the Ig-like domain since both C5 and C5b were equally recognized by an anti-PD1 antibody. Similar results were obtained for C6 and C6b recognized by an anti-SLAM antibody. Taken together, these findings suggest that domains can be inserted in the elbow with additional linkers that distance such domains from the main antibody scaffold. Without being bound to any theory, it is assumed that the linkers can provide enhanced flexibility of the insert.

Example 5: Design of an Antibody Containing a Molecular Tag Inserted in the Elbow Region

[0321] To investigate the possibility to use a tag inserted into the elbow region of an antibody for antibody detection, one construct (named C9) was designed, in which a twin Strep-tag was inserted into the elbow region of the heavy chain of scaffold antibody C1b (cf. Example 1). The light chain of the construct was not modified in comparison to the scaffold antibody. The construct was finally expressed as monoclonal antibody (heavy and light chains).

[0322] Construct C9 is shown schematically in FIG. 2. In construct C9 a twin Strep-tag (2XST; SEQ ID NO: 20) was inserted into the elbow region of a recombinant monospecific antibody C1b (see above). Construct C9 (complete heavy chain: SEQ ID NO: 65, complete light chain: SEQ ID NO: 63) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain (VH3-20); the expression product of a D (diversity) gene segment of a heavy chain variable domain (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain (JH3); the expression product of a twin Strep-tag (2XST); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain and the expression product of a J (Joining) gene segment element of a light chain variable domain; the expression product of a C (constant) gene segment of a light chain kappa constant region.

Example 6: Molecular Tags can be Inserted in the Elbow Region of Antibodies Resulting in Functional Antibodies

[0323] The antibody construct C9 described in Example 5 was produced recombinantly by transient transfection. To this end, antibody heavy and light chains were cloned into human IgG1 and Igx expression vectors and expressed by transient transfection of Expi293F Cells (ThermoFisher Scientific) using polyethylenimine (PEI). Cell lines were routinely tested for mycoplasma contamination.

[0324] Next, the antibody construct C9 and control antibody C1b (see Example 1) were tested for recognition by Strep-tag-specific Strep-tactin molecule by ELISA. Briefly, total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as a standard. To test specific recognition of the antibody construct, ELISA plates were coated with 10 g ml.sup.1 of an anti-human IgG antibody (SouthernBiotech), plates were blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies, followed by AP-conjugated Strep-Tactin (Iba Lifesciences). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm.

[0325] Results are shown in FIG. 7. Antibody construct C9 carrying a Strep-tag was efficiently and specifically recognized by Strep-tag-specific Strep-tactin, whereas the control C1b antibody that does not carry Strep-tag was not recognized. These results suggest that the elbow is permissive for insertion of molecular tags that allow recognition and tracing of an antibody.

Example 7: Design of Antibodies Containing a Single-Chain-Antibody Variable Domain (VHH) or a Single-Chain Variable Fragment (ScFv) Inserted in the Elbow Region

[0326] To investigate whether insertion of (additional) functional domains other than Ig-like domains into the elbow region results in functional multispecific antibodies, four new constructs were designed (C9-C12). In the four new constructs C9-C12, a single-chain-antibody variable domain (VHH) or a single-chain variable fragment (ScFv), specific for tetanus toxoid (TT) or for the fusion (F) protein of the respiratory syncytial virus (RSV), respectively, were inserted into the elbow region of an antibody specific for influenza H1 hemagglutinin (FI174; Pappas, L., et al. Rapid development of broadly influenza neutralizing antibodies through redundant mutations. Nature 516, 418-422 (2014); VH: SEQ ID NO: 10, VI: SEQ ID NO: 11, heavy chain constant region: SEQ ID NO: 3, light kappa chain constant region: SEQ ID NO: 12/SEQ ID NO: 4). The light chain of the constructs was not modified in comparison to the scaffold antibody FI1174. All construct were finally expressed as monoclonal antibodies (heavy and light chains).

[0327] The following variants were produced and are shown schematically in FIG. 8: [0328] 1. In construct C10 an anti-T VHH (T3-VHH; Rossotti, M. A., et al. Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain. mAbs 7, 820-828 (2015); SEQ ID NO: 16) was inserted into the elbow region of F1174. Construct C10 (complete heavy chain: SEQ ID NO: 66, complete light chain: SEQ ID NO: 67) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of FI174 (VH); the expression product of a D (diversity) gene segment of a heavy chain variable domain of F1174 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of FI174 (JH); the expression product of an anti-TT VHH (T3-VHH; Rossotti, M. A., et al. Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain. mAbs 7, 820-828 (2015)); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of FI74 and the expression product of a J (Joining) gene segment element of a light chain variable domain of FI174; the expression product of a C (constant) gene segment of a light chain constant region. [0329] 2. In construct C11 an anti-TT ScFv (TT39.7-ScFv; SEQ ID NO: 17) was inserted into the elbow region of FI74. Construct C11 (complete heavy chain: SEQ ID NO: 68, complete light chain: SEQ ID NO: 67) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of F1174 (VH); the expression product of a D (diversity) gene segment of a heavy chain variable domain of F1174 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of F1174 (JH); the expression product of an anti-ScFv (TT39.7-ScFv); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of FI174 and the expression product of a J (Joining) gene segment element of a light chain variable domain of FI174; the expression product of a C (constant) gene segment of a light chain constant region. [0330] 3. In construct C12 an anti-RSV F protein VHH (F4-VHH; Rossey, I., et al. Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state. Nature communications 8, 14158 (2017); SEQ ID NO: 18) was inserted into the elbow region of F1174. Construct C12 (complete heavy chain: SEQ ID NO: 69, complete light chain: SEQ ID NO: 67) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of FI1174 (VH): the expression product of a D (diversity) gene segment of a heavy chain variable domain of FI174 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of FI174 (H); the expression product of an anti-RSV F protein VHH (F4-VHH; Rossey, I., et al. Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state. Nature communications 8, 14158 (2017)); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of FI174 and the expression product of a J (Joining) gene segment element of a light chain variable domain of F1174; the expression product of a C (constant) gene segment of a light chain constant region. [0331] 4. In construct C13 an anti-RSV F protein ScFv (MPE8-ScFv; Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013); SEQ ID NO: 19) was inserted into the elbow region of FI174. Construct C13 (complete heavy chain: SEQ ID NO: 70, complete light chain: SEQ ID NO: 67) is formed by (in this order from N- to C-terminus): the expression product of a V (variable) gene segment of a heavy chain variable domain of FI74 (VH); the expression product of a D (diversity) gene segment of a heavy chain variable domain of F1174 (D); the expression product of a J (Joining) gene segment element of a heavy chain variable domain of F1174 (H); the expression product of an anti-RSV F protein ScFv (MPE8-ScFv; Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)); the expression product of a C (constant) gene segment of a heavy chain constant region (IgG1 isotype); and on a separate chain: the expression product of a V (variable) gene segment of a light chain variable domain of FI74 and the expression product of a J (Joining) gene segment element of a light chain variable domain of FI174; the expression product of a C (constant) gene segment of a light chain constant region.

Example 8: VHH and ScFv can be Inserted in the Elbow Region Resulting in Functional Bispecific Antibodies

[0332] The four new antibody constructs described in Example 4 were produced recombinantly by transient transfection. To this end, antibody heavy and light chains were cloned into human IgG1, Ig and Ig expression vectors and expressed by transient transfection of Expi293F Cells (ThermoFisher Scientific) using polyethylenimine (PEI). The constructs were tested for dual binding to (i) H1 and (ii) either TT or RSV F protein by surface plasmon resonance (SPR).

[0333] In a first experiment, constructs C10 and C11 (with TT-specific VHH or scFv in elbow region of FI74) were analyzed. To this end, protein A was used to capture the constructs followed by co-injection of the analytes (H1 as first analyte immediately followed by TT). As controls TT-specific antibody TT107 and the H1-specific antibody FI174 were used. Briefly, protein A (25 g/ml) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized onto an ethyl(dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) pre-activatedProteOnsensor chip (Bio-Rad) through amine coupling; unreacted groups were blocked by injection of 1M ethanolamine HCl. HEPES buffered saline (HBS) (10 mMHEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20) was used as a running buffer. All injections were made at a flowrate of 100 l/min. Monoclonal antibodies were diluted in HBS to 10 nM and injected for 240 s onto the Protein A-coated chip for capturing, followed by co-injection of H1 California hemagglutinin (50 nM) immediately followed by tetanus toxoid (50 nM) for total 110 s. One channel of the chip was injected with HBS and used as reference for the analysis. Each binding interaction of the monoclonal antibodies to H1 and TT was assessed using a ProteONXPR36 instrument (Bio-Rad) and the data were processed with ProteOn Manager software. Results are shown in FIG. 9. Constructs C10 and C11 (carrying a TT-specific domain (VHH or ScFv)) bound both H1 and TT, while control antibodies FI174 and TT107 recognize only H1 or only TT, respectively.

[0334] In a second experiment, constructs C12 and C13 (with RSV F-protein-specific VHH or scFv in elbow region of F1174) were analyzed. To this end, protein A was used to capture the constructs followed by co-injection of the analytes (H1 as first analyte immediately followed by RSV F protein). As controls RSV F protein-specific antibody MPE8 and the H1-specific antibody F1174 were used. Briefly, protein A (25 g/ml) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized onto an ethyl(dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) pre-activatedProteOnsensor chip (Bio-Rad) through amine coupling; unreacted groups were blocked by injection of 1M ethanolamine HC. HEPES buffered saline (HBS) (10 mMHEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20) was used as a running buffer. All injections were made at a flowrate of 100 l/min. Monoclonal antibodies were diluted in HBS to 10 nM and injected for 240 s onto the Protein A-coated chip for capturing, followed by co-injection of H1 California hemagglutinin (50 nM) immediately followed by RSV F protein (50 nM) for total 110 s. One channel of the chip was injected with HBS and used as reference for the analysis. Each binding interaction of the monoclonal antibodies to H1 and RSV F protein was assessed using a ProteONXPR36 instrument (Bio-Rad) and the data were processed with ProteOn Manager software. Results are shown in FIG. 10. Constructs C12 and C13 (carrying a RSV F protein-specific domain (VHH or ScFv)) bound both H1 and RSV F protein, while control antibodies FI174 and MPE8 recognize only H1 or only RSV F protein, respectively.

[0335] In summary, the data show that insertion of VHH or ScFv domains into the elbow region of FI174 did not affect binding to H1 hemagglutin, indicating that this site is permissive for insertions of different domains without affecting the original antibody specificity, as already shown above for the Ig-like domains. Dual simultaneous recognition of the two different specific antigens by the VDJ region of the antibody scaffold and the VHH or ScFv domains inserted in the elbow indicates that it is possible to generate functional and bispecific antibodies carrying different types of inserts in the elbow.

TABLE-US-00001 TABLEOFSEQUENCESANDSEQIDNUMBERS(SEQUENCELISTING): SEQIDNO Sequence Remarks SEQIDNO:1 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV GCE536VHaa WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRV TVTTDASTNTVDMELRSLRSEDTAVYYCARGPRSKP PYLYFALDVWGQGTAVTVSS SEQIDNO:2 EIVLTQSPGTLSLSPGETAILSCRASQSVSSSLLAWYQ GCE536VLaa QKPGQAPRLLIYGASNRATGIRGRFSGSGSGTDFTL TISRLEPEDFVLYYCQHYGSRVTFGQGTKLEIK SEQIDNO:3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV HumanHCIgG1 TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS constantregionaa SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:4 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA HumanLCkappa KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL constantregionaa TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:5 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGI C1VHaa HWVRQAPGKGLEWLSLIENHGRKIYYAESVKGRIT VSRDNFKNVAYLEMYRLSTEDTAIYYCARNDGLGR YTDAGGTHRTAYLDYWGRGTLVTVSS SEQIDNO:6 SYEVTQPPSVSVSPGQAARITCSGDELPRTDISWYQ C1VLaa QTSGQAPVLVIYEGTKRPSGIPERFSGSVSGAMATL MISEAQLEDEGDYYCFSIDTSGNHGGAFGTGTKLT VL SEQIDNO:7 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA HumanLClambda VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYL constantregionaa SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQIDNO:8 DVQLVESGGGVVRPGVSLRLSCVASGESEKNYDM C1bVHaa AWVRQVPGKGLEWVCGINWNGSLRGYADSVKG RFLISRDHAKDSLYLQMSRLRAEDTALYYCARDPGY NTGRDHPYDLWGQGTMVTVSS SEQIDNO:9 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWY C1bVLaa QQKPGKAPKLLIYKASSLGSGVPSRFSGSGSGTQFT LTISSLQPDDFATYYCQQYNNYPYTFGQGTKLEIK SEQIDNO:10 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALS FI174VHaa WVRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRV TINADESTSTVYLELSSLTSEDTAIYYCAGSATYYESRF DYWGQGTLVTVSS SEQIDNO:11 EIVLTQSPGTLSLSPGARATLSCRASQSVSSSSLAWY FI174VLaa QQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFT LTISRLEPEDFAVYYCHQYGDSRKTFGQGTKVEIK SEQIDNO:12 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL unmutatedLAIR1 ERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAG fragmentaa PYRCIYYKPPKWSEQSDYLELLVK SEQIDNO:13 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL mutatedLAIR1 ERERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNA fragmentaa GLFRCIYYKSRKWSEQSDYLELVVK SEQIDNO:14 DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSE PD-1fragmentaa SFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRF RVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAP KAQIKESLRAELRVT SEQIDNO:15 EQVSTPEIKVLNKTQENGTCTLILGCTVEKGDHVAY SLAMfragmentaa SWSEKAGTHPLNPANSSHLLSLTLGPQHADNIYICT VSNPISNNSQTFSPWPGCRTDPS SEQIDNO:16 MAQVQLVESGGGLVQAGGSLTLSCAASGSTSRSY T3-VHHaa ALGWFRQAPGKEREFVAHVGQTAEFAQGRFTISR DFAKNTVSLQMNDLKSDDTAIYYCVASNRGWSPS RVSYWGQGTQVTVSS SEQIDNO:17 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSRVGVG TT39.7-scFvaa WIRQPPGKALEWLSLIYWDDEKHYSPSLKNRVTISK DSSKNQVVLTLTDMDPVDTGTYYCAHRGVDTSG WGFDYWGQGALVTVSSGGGGSGGGGSGGGGS QSALTQPASVSGSPGQSITISCSGAGSDVGGHNFV SWYQQYPGKAPKLMIYDVKNRPSGVSYRFSGSKSG YTASLTISGLQAEDEATYFCSSYSSSSTLIIFGGGTRLT VL SEQIDNO:18 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIG F4-VHHaa WFRQAPGKEREAVSCISGSSGSTYYPDSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCATIRSSSWGG CVHYGMDYWGKGTQVTVSS SEQIDNO:19 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMN MPE8-scFvaa WVRQAPGKGLEWVSSISASSSYSDYADSAKGRFTIS RDNAKTSLFLQMNSLRAEDTAIYFCARARATGYSSI TPYFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQ SVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVH WYQQLPGTAPKLLIYDNNNRPSGVPDRFSASKSGT SASLAITGLQAEDEADYYCQSYDRNLSGVFGTGTK VTVL SEQIDNO:20 SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK twinStrepTagaa SEQIDNO:21 GLNDIFEAQKIEWHE AviTag SEQIDNO:22 KRRWKKNFIAVSAANRFKKISSSGAL Calmodulin-tag SEQIDNO:23 EEEEEE polyglutamatetag SEQIDNO:24 GAPVPYPDPLEPR E-tag SEQIDNO:25 DYKDDDDK FLAG-tag SEQIDNO:26 YPYDVPDYA HA-tag SEQIDNO:27 HHHHHH His-tag SEQIDNO:28 EQKLISEEDL Myc-tag SEQIDNO:29 TKENPRSNQEESYDDNES NE-tag SEQIDNO:30 KETAAAKFERQHMDS S-tag SEQIDNO:31 MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQ SBP-tag GQREP SEQIDNO:32 SLAELLNAGLGGS Softag1 SEQIDNO:33 TQDPSRVG Softag3 SEQIDNO:34 WSHPQFEK Strep-tag SEQIDNO:35 CCPGCC TCtag SEQIDNO:36 GKPIPNPLLGLDST V5tag SEQIDNO:37 YTDIEMNRLGK VSV-tag SEQIDNO:38 DLYDDDDK Xpresstag SEQIDNO:39 TDKDMTITFTNKKDAE Isopeptag SEQIDNO:40 AHIVMVDAYKPTK SpyTag SEQIDNO:41 KLGDIEFIKVNK SnoopTag SEQIDNO:42 EVHTNQDPLD Ty1tag SEQIDNO:43 GGGGS linker SEQIDNO:44 GGGGSGGGGS linker SEQIDNO:45 GGGGSGGGGSGGGGS linker SEQIDNO:46 GGGGSGGGGSGGGGSGGGGS linker SEQIDNO:47 GGGGSGGGGSGGGGSGGGGSGGGGS linker SEQIDNO:48 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker SEQIDNO:49 FVGPPSPFLTSLHLS linker SEQIDNO:50 GAASEAPGQGLAEPV linker SEQIDNO:51 QGFTSVMAPFLPLLT linker SEQIDNO:52 GEYTGGSLCATLMSM linker SEQIDNO:53 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGI C2heavychainaa HWVRQAPGKGLEWLSLIENHGRKIYYAESVKGRITV SRDNFKNVAYLEMYRLSTEDTAIYYCARNDGLGRY TDAGGTHRTAYLDYWGRGTLVTVSSEDLPRPSISAE PGTVIPLGSHVTFVCRGPVGVQTFRLERERNYLYSD TEDVSQTSPSESEARFRIDSVNAGNAGLFRCIYYKSR KWSEQSDYLELVVKASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCS VMHEALHNHYTQKSLSLSPGK SEQIDNO:54 SYEVTQPPSVSVSPGQAARITCSGDELPRTDISWYQ C2/C3lightchainaa QTSGQAPVLVIYEGTKRPSGIPERFSGSVSGAMATL MISEAQLEDEGDYYCFSIDTSGNHGGAFGTGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYP GAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC S SEQIDNO:55 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGI C3heavychainaa HWVRQAPGKGLEWLSLIENHGRKIYYAESVKGRIT VSRDNFKNVAYLEMYRLSTEDTAIYYCARNDGLGR YTDAGGTHRTAYLDYWGRGTLVTVSSEDLPRPSIS AEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYY KPPKWSEQSDYLELLVKASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKIIPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:56 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV C4heavychainaa WVRQAPGQGLEWMATISPGDVNTSYEQRFQGR VTVTTDASTNTVDMELRSLRSEDTAVYYCARGPRS KPPYLYFALDVWGQGTAVTVSSEDLPRPSISAEPGT VIPLGSFIVTFVCRGPVGVQTFRLERESRSTYNDTED VSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKW SEQSDYLELLVKASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:57 EIVLTQSPGTLSLSPGETAILSCRASQSVSSSLLAWY C4/C5/C6lightchain QQKPGQAPRLLIYGASNRATGIRGRFSGSGSGTDF aa TLTISRLEPEDEVLYYCQHYGSRVTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC SEQIDNO:58 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV C5heavychainaa WVRQAPGQGLEWMATISPGDVNTSYEQRFQGR VTVTTDASTNTVDMELRSLRSEDTAVYYCARGPRS KPPYLYFALDVWGQGTAVTVSSDSPDRPWNPPTF SPALLVVTEGDNATFTCSESNTSESFVLNWYRMSPS NQTDKLAAFPEDRSQPGQDCRERVTQLPNGRDF HMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAEL RVTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYE PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEA LHNHYTQKSLSLSPGK SEQIDNO:59 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV C5bheavychainaa WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRV TVTTDASTNTVDMELRSLRSEDTAVYYCARGPRSKP PYLYFALDVWGQGTAVTVSSFVGPPSPFLTSLHLSD SPDRPWNPPTESPALLVVTEGDNATETCSFSNTSESF VLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKA QIKESLRAELRVTGAASEAPGQGLAEPVASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKIIPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:60 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV C6heavychainaa WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRV TVTTDASTNTVDMELRSLRSEDTAVYYCARGPRSKP PYLYFALDVWGQGTAVTVSSFVGPPSPFLTSLHLSD SPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESF VLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKA QIKESLRAELRVTGAASEAPGQGLAEPVASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKS RWQQGNVESCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:61 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV C6bheavychainaa WVRQAPGQGLEWMATISPGDVNTSYEQRFQGR VTVTTDASTNTVDMELRSLRSEDTAVYYCARGPRS KPPYLYFALDVWGQGTAVTVSSQGFTSVMAPFLPL LTEQVSTPEIKVLNKTQENGTCTLILGCTVEKGDHV AYSWSEKAGTHPLNPANSSHLLSLTLGPQHADNIY ICTVSNPISNNSQTFSPWPGCRTDPSGEYTGGSLC ATLMSMASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMH EALHNHYTQKSLSLSPGK SEQIDNO:62 DVQLVESGGGVVRPGVSLRLSCVASGESEKNYDM C7heavychainaa AWVRQVPGKGLEWVCGINWNGSLRGYADSVKG RFLISRDHAKDSLYLQMSRLRAEDTALYYCARDPG YNTGRDHPYDLWGQGTMVTVSSDSPDRPWNPP TESPALLVVTEGDNATFTCSFSNTSESEVLNWYRMS PSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK SEQIDNO:63 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAW C7/C8/C9lightchain YQQKPGKAPKWYKASSLGSGVPSRFSGSGSGTQF aa TLTISSLQPDDFATYYCQQYNNYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C SEQIDNO:64 DVQLVESGGGVVRPGVSLRLSCVASGESEKNYDMA C8heavychainaa WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRF LISRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNT GRDHPYDLWGQGTMVTVSSEQVSTPEIKVLNKTQ ENGTCTLILGCTVEKGDHVAYSWSEKAGTHPLNPA NSSHLLSLTLGPQHADNIYICTVSNPISNNSQTESPW PGCRTDPSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVIVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPGK SEQIDNO:65 DVQLVESGGGVVRPGVSLRLSCVASGESEKNYDM C9heavychainaa AWVRQVPGKGLEWVCGINWNGSLRGYADSVKG RFLISRDHAKDSLYLQMSRLRAEDTALYYCARDPG YNTGRDHPYDLWGQGTMVTVSSSAWSHPQFEK GGGSGGGSGGSAWSHPQFEKASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:66 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALS C10heavychainaa WVRQAPGQGLEWMGGIIAIFGTTNYAQKEQGRV TINADESTSTVYLELSSLTSEDTAIYYCAGSATYYESR FDYWGQGTLVTVSSMAQVQLVESGGGLVQAGG SLTLSCAASGSTSRSYALGWFRQAPGKEREFVAHV GQTAEFAQGRFTISRDFAKNTVSLQMNDLKSDDT AIYYCVASNRGWSPSRVSYWGQGTQVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQIDNO:67 EIVLTQSPGTLSLSPGARATLSCRASQSVSSSSLAWY C10/C11/C12/C13 QQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDF lightchainaa TLTISRLEPEDFAVYYCHQYGDSRKTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C SEQIDNO:68 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALS C11heavychainaa WVRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRV TINADESTSTVYLELSSLTSEDTANYCAGSATYYESR FDYWGQGTLVTVSSQITLKESGPTLVKPTQTLTLTC TFSGFSLSTSRVGVGWIRQPPGKALEWLSLIYWDD EKHYSPSLKNRVTISKDSSKNQVVLTLTDMDPVDT GTYYCAHRGVDTSGWGFDYWGQGALVTVSSGG GGSGGGGSGGGGSQSALTQPASVSGSPGQSITIS CSGAGSDVGGHNEVSWYQQYPGKAPKLMIYDV KNRPSGVSYRFSGSKSGYTASLTISGLQAEDEATYF CSSYSSSSTLIIFGGGTRLTVLASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:69 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALSW C12heavychainaa VRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRVTI NADESTSTVYLELSSLTSEDTAIYYCAGSATYYESRFD YWGQGTLVTVSSQVQLQESGGGLVQPGGSLRLSC AASGFTLDYYYIGWFRQAPGKEREAVSCISGSSGST YYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCATIRSSSWGGCVHYGMDYWGKGTQVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQIDNO:70 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALS C13heavychainaa WVRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRV TINADESTSTVYLELSSLTSEDTAIYYCAGSATYYESR FDYWGQGTLVTVSSEVQLVESGGGLVKPGGSLRL SCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASS SYSDYADSAKGRFTISRDNAKTSLFLQMNSLRAEDT AIYFCARARATGYSSITPYFDIWGQGTLVTVSSGG GGSGGGGSGGGGSQSVVTQPPSVSGAPGQRVTI SCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNN NRPSGVPDRFSASKSGTSASLAITGLQAEDEADYYC QSYDRNLSGVEGTGTKVTVLASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:71 QVQLQESGPGLVKPSGUSLTCAVSGGSISSSNW MGD.sup.UCAVHaa WSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRV TISVDKSKNQFSLKLSSVTAADTAVYYCARASPLKS QRDTEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGV QTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVS EGNAGPYRCIYYKPPKWSEQSDYLELLVKGEDVTW ALPQSQLDPRACPQGELPISTDIYYMDVWGKGTT VTVSS SEQIDNO:72 AIRMTQSPSSFSASTGDRVTITCRASQGISSYLAWY MGD.sup.UCAVLaa QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFT LTISCLQSEDFATYYCQQYYSYPPDFGQGTRLEIK SEQIDNO:73 QVQLIQSGGGLVKPGGSLRLSCAASGFTFSDYYMS TT107VHaa WIRQVPGKGLEWISVISATTGYTDYADSVKGRFTIS RDNAKNSVFLQMNSLRVDDMAVYYCAREVLGTA WFDYWGQGTLVTISS SEQIDNO:74 EIVLTQSPGTLSLSPGERATLSCRASQSVTSNYLAWY TT107VLaa QQKPGQAPRLLIYGVSRRATGIPDRFSGSGSGTDF ALTISRLEPEDFAVYYCQQYRSSPRTFGPGTKVEFK SEQIDNO:75 EVQLVESGGGVVRPGESLRLSCAASGFIFNDEGMN MGJ1VH WVRQPPGRGLEWVAGIKWRGGGVALVPSVTGRF TISGDNDKNSLYLQMTSLRDEDTAVYYCARDSGER GGRGHAFDLWGQGTMVTISAEDLPRPSISAEPGTV IPLGSHVITVCRGPVGVHTFRLERESRSTYNETEDVS QASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK SEQIDNO:76 EVQVVESGGRVARPGGSLRLSCAASGFHLDDYDM MGJ2VH SWVRQPPGKGLEWVAGINWNGGRTGYADSVKG RLTISRDNAKKFLYLEMKSLRAEDTALYYCARDPGYS SGRRNALDIWGQGTMVTVSLEDLPRPSISAEPGTVI PLGSHVTFVCRGPVGVQTFRLERESRFTYNDTEDVS QASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK SEQIDNO:77 EVQLVQSGGGVVRPGGFLRLSCAASGFTFENYAVA MGJ3VH WVRQVAGKGLEWLCVINWDAGTTNYADSVKGRF TISRDIVKNSLVLEMSSLRAEDTALYYCARDPVYGSD RGDVEDMWGQGTVVTVSSDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQA SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:78 DVQLVESGGGVVRPGVSLRLSCVASGESEKNYDMA MGJ5VH WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRF LISRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNT GRDHPYDLWGQGTMVTVSSEDLPRPSISAEPGTVI PLGSHVTFVCRGPVGVQTFRLERESRSTYNETEDVS QVSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQ SDYLELLVK SEQIDNO:79 EVQLVESGGRVVRPGESLRLSCEVSGVSINDYDMS MMJ1VH WVRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLTGDDTAFYYCVRDPGESS GRGHIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQV SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:80 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMS MMJ2VH WVRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGEDTALYYCVRDPGDTS GRGHIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIP LGSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQ VSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQS DYLELLVK SEQIDNO:81 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMS MMJ5VH WVRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDNGKNSLYLQMNSLRGEDTALYYCVRDPGDRS GRGHIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQV SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:82 EVQLVESGGGVVRPGESLRLSCEVSGVSINDYDMS MMJ6VH WVRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGEDTALYYCVRDPGDSS GRGQIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQV SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:83 EVQLVESGGGVVRPGESLRLSCEVSGVSINDYDMS MMJ7VH WVRQRLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGEDTALYYCVRDPGESS GRGHIFNIWGQGTMVTISLEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQV SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:84 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMS MMJ8VH WVRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGEDTALYYCVRDPGDTS GRGHIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIP LGSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQ VSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQS DYLELLVK SEQIDNO:85 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMS MMJ10VH WVRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGEDTALYYCVRDPGDTS GRGHIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIP LGSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQ VSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQS DYLELLVK SEQIDNO:86 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMS MMJ16VH WVRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGEDTALYYCVRDPGDTS GRGHIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIP LGSHVTEVCRGPVGVQTFRLERESRSTYNETEDVSQ VSPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQS DYLELLGK SEQIDNO:87 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMS MMJ23VH WVRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDNGKNSLYLQMNSLRGEDTALYYCVRDPGDRS GRGHIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQV SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:88 EVQLVESGGGVVRPGESLRLSCEVSGVSINDYDMS MMJ25VH WVRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFT ISRDHAKNSLYLQMNSLRGADTALYYCVRDPGDSS GRGHIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQV SPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSD YLELLVK SEQIDNO:89 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGI MGM1VH HWVRQAPGKGLEWLSLIENHGRKIYYAESVKGRITV SRDNFKNVAYLEMYRLSTEDTAIYYCARNDGLGRY TDAGGTHRTAYLDYWGRGTLVTVSSEDLPRPSISAE PGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYND TEDVSQASPSESEARFRIDSVSEGNAGPYRCVYYKPP KWSEQSDYLDLLVK SEQIDNO:90 QVQLVESGGDVVQPGGSLRLSCAVSGEKENIYDIH MGM3VH WVRQAPGKGLEWVSFIRHDGNNQEYADSVKGRF TISRDNFKNIIDLQMHSLRTEDTALYYCATNQGSGG SDDTWETNRSAFFPHWGQGTLVTVSSDLPRPSISAE PGTVIPLGSHVTFVCRGPVGVQTFRLERESRSIYNDT EDVSQASPSESEARFRIDSVSEGNAGPYRCVYYKPPK WSEESDYLELLVK SEQIDNO:91 QVQLVESGGGVVQPGGSLRLSCEVSGFRESTYGIH MGM4VH WARQAPGKGLEWVAFIRYDGNNKSYADSVKGRFT ISRDNSKNTLYLQMNSLRIEDTAVYYCAKNQASGG YDDTWGTYRSAYLDYWGQGTLVTVSSEDLPRPSIS AEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYN DTEDVSQASPSESEARFRIDSVSEGNAGPYRCVYYKP PKWSEESDSLELLVK SEQIDNO:92 QVQLVESGGGVVQPGGSLRLSCKMSGEKESAFGIH MGM5VH WVRQAPGKGLEWVAFVRYDGGDKYYADSVKGRF TISRDNSKNTVHLQLNSLKPADTAVYYCAKNQPSG QSDDTWGTSLSAYLDYWGQGTQVSVSPEDLPRPSI SAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTY NDTEDVSQASPSESEARFRIDSVSEGNAGPYRCVYY KPPKWSEQSDYLELLVK SEQIDNO:93 EAQVVDHGNRGRARDLEDIKKRRARDLEYEDLPRP MGB2VH SISAEPGTVIPLGSRVTFVCRGPVGVQTFRLERESRSK YNETEDVSQASPSESEARFRIDSVSEGNAGPYRCIYY KPPKWSEHSDFLELLVK SEQIDNO:94 VAEVEEHINKRRARDLEYEDLPRPSISAEPGTVIPLGS MGB43VH HVTFVCRGPVGVQTFRLERESRSRYNETEDVSQTSP SESEARFRIDSVSEGNAGPYRCLYYKTPKWSEQSDFL ELLVK SEQIDNO:95 EAEVVEHVNKRRARALEYEDLPRPSISAEPGTVIPLGS MGB47VH HVTFVCRGPVGVQTFRLERESRSRYTETEDVSQTSPS ESEARFRIDSVSEGNAGPYRCLYYKPPKWSEQSDFLE LLVK SEQIDNO:96 DFQMTQSPSTLSASVGDRVTITCRASQNVNTWLA MGJ1VL WYQQAAGKAPKLLIYEASTLQSGVPSRFRGGGSGT EFTLTITSLQPEDFATYYCHQYKSHPFTFGPGTKVDV R SEQIDNO:97 DIQMTQSPSTLSASVGDRVTITCRASQTISSWLAWY MGJ2VL QQKPGKAPKFLIYKASFLENGVPSRFSGSESGTEFTLT INSLQPDDFATYYCQQYKSYPFTFGPGTKVEIK SEQIDNO:98 DIQMTQSPSTLSASVGDRVTFTCGASQSITDCLAW MGJ3VL YQQKPGKDPKLLIYKASRLEAGVPARFSASGSGTEFT FTIRSMQPEDFATYYCQQCYSYPFTFGPGTKVDLK SEQIDNO:99 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWY MGJ5VL QQKPGKAPKLLIYKASSLGSGVPSRFSGSGSGTQFTL TISSLQPDDFATYYCQQYNNYPYTFGQGTKLEIK SEQIDNO:100 DIQMTQSPSTVSASIGDRVTITCRASQIIERSLAWYQ MMJ1VL QKPGKSPKALIYKTSNLEDGVPSRFSGSGSGTDFTLT VSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:101 DIQMTQSPSTLSASIGDRVTITCRASQVIDRSLAWF MMJ5VL QQKPGKSPRPLIYKASTLEGGVPSRFSGSGSGTDFTL TVSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:102 DIQMTQSPSTLSASIGDRVTITCRASQIIHRSLAWYQ MMJ6VL QKPGKSPRALIYKASNLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFAMYYCQQYDTYPFTFGPGTTVFLR SEQIDNO:103 DIQMTQSPSTLSASIGDRVTITCRASQSIDRSLAWY MMJ7VL QQKPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFT LTVSSLQPDDFADYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:104 DIQMTQSPSTLSASIGDRVTITCRASQIIDRSLAWYQ MMJ8VL QKPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:105 DIQMTQSPSTLSASIGDRVTITCRASQIIDRSLAWYQ MMJ10VL QKPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:106 DIQMTQSPSTLSASIGDRVTITCRASQIIDRSLAWYQ MMJ16VL QKPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:107 DIQMTQSPSTLSASIGDRVTITCRASQVIDRSLAWF MMJ23VL QQKPGKSPRPLIYKASTLEGGVPSRESGSGSGTDFTL TVSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:108 DIQMTQSPSTLSASIGDRVTITCRASQNIDRSLAWY MMJ25VL QQKPGKSPKALIYKASNLEDGVPSRFSGSGSGTDFT LTVSSLQPDDFALYYCQQYDTYPFTFGPGTTVTLR SEQIDNO:109 SYEVTQPPSVSVSPGQAARITCSGDELPRTDISWYQ MGM1VL QTSGQAPVLVIYEGTKRPSGIPERFSGSVSGAMATL MISEAQLEDEGDYYCFSIDTSGNHGGAFGTGTKLT VL SEQIDNO:110 SYELIQPPSXSVSPGQTARITCSGEPLPRTSTSWYRQK MGM3VL SGQAPVLIIYEVSKRPSGIPERXSGSNTGTKATLFIVG AQVEDEGDYYCYSTNTSGGSRGAFGTGTSLTVL SEQIDNO:111 SYELTQPPSVSVSPGQTARITCSGDAVPNTYTYWYQ MGM4VL QKSGQAPVLVIYEDSKRPSGIPERESGSSSGTMATFII SGAQVEDEADYYCYSTDTSDDHRGAFGTGTKVTV L SEQIDNO:112 SYELTQFPSVSVSPGQTARITCSGDALPRTFIYWYQ MGM5VL QKSRQAPVVVIYEDVKRPSGIPERFSGSISGTQATLII TGAQVEDEADYYCYSTDTNNTHRGAFGTGTKVTV L SEQIDNO:113 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVHTFRL MGJ1LAIR1 ERESRSTYNETEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:114 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGJ2LAIR1 ERESRFTYNDTEDVSQASPSESEARFRIDSVSEGNAG PYRCIYYKPPKWSEQSDYLELLVK SEQIDNO:115 DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE MGJ3LAIR1 RESRSTYNETEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:116 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGJ5LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:117 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ1LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:118 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ2LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:119 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ5LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:120 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ6LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:121 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ7LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:122 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ8LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:123 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ10LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:124 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ16LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLGK SEQIDNO:125 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ23LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:126 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MMJ25LAIR1 ERESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK SEQIDNO:127 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGM1LAIR1 ERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAG PYRCVYYKPPKWSEQSDYLDLLVK SEQIDNO:128 DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE MGM3LAIR1 RESRSIYNDTEDVSQASPSESEARFRIDSVSEGNAGP YRCVYYKPPKWSEESDYLELLVK SEQIDNO:129 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGM4LAIR1 ERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAG PYRCVYYKPPKWSEESDSLELLVK SEQIDNO:130 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGM5LAIR1 ERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAG PYRCVYYKPPKWSEQSDYLELLVK SEQIDNO:131 EDLPRPSISAEPGTVIPLGSRVTFVCRGPVGVQTFRLE MGB2LAIR1 RESRSKYNETEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEHSDFLELLVK SEQIDNO:132 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGB43LAIR1 ERESRSRYNETEDVSQTSPSESEARFRIDSVSEGNAGP YRCLYYKTPKWSEQSDFLELLVK SEQIDNO:133 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL MGB47LAIR1 ERESRSRYTETEDVSQTSPSESEARFRIDSVSEGNAGP YRCLYYKPPKWSEQSDFLELLVK