RU2796254C2 - Antibodies containing polypeptide integrated into frame region 3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody construct is proposed to improve the pharmacokinetics of an insert polypeptide, containing a frame antibody that contains a variable (V) domain and an insert polypeptide. The insert polypeptide is located in the framework region 3 of the V domain. Nucleic acids, expression vectors, cloning vectors, and host cells for producing the said antibody construct are also provided.

EFFECT: invention provides a new format of engineered antibody which demonstrates improved pharmacokinetics, for example, increased serum half-life.

32 cl, 24 dwg, 7 tbl, 15 ex

Description

FIELD OF THE INVENTION

The present invention relates to antibodies containing a variable (V) domain and an insert polypeptide in the framework region 3 of the V domain. Such antibodies may provide improved functionality and/or pharmacokinetics of the insert polypeptide.

STATE OF THE ART TO which THE INVENTION RELATES

The high specificity and affinity of antibodies make them ideal diagnostic and therapeutic agents. Advances in recombinant antibody technology have led to the production of antibody fragments such as Fv, Fab, Fab' and F(ab') ₂ fragments. These smaller molecules retain the antigen-binding activity of whole antibodies and also show improved tissue penetration and pharmacokinetic properties compared to whole immunoglobulin molecules. Indeed, antibody fragments have proven to be versatile therapeutic agents. While such fragments have proven to show a number of advantages over whole immunoglobulins, they also have an increased serum clearance rate as they lack the Fc domain that provides a long in vivo half-life (Medasan et al., 1997, J. Immunol 158:2211-2217). Previous approaches to improve the serum half-life of antibody molecules include conjugation with agents or domains that interact with the FcRn receptor, such as human serum albumin (HSA). PEG has also been shown to increase serum half-life.

Antibodies with dual specificity have previously been described, ie. that bind to two different antigens (for a review, see Segal et al., 1999, Curr. Opin. Immunol. 11:558-562; Pluckthun & Pack, 1997, Immunotechnology, 3:83-105; Fischer and Leger, 2007 , Pathobiology, 74: 3-14). Previous approaches to obtain heterobispecific antibody-based molecules have mainly used chemical crosslinking or protein engineering methods. The disadvantage of chemical crosslinking is the low yield of hetero- and homodimer formation and the need for their subsequent chromatographic separation. Protein engineering approaches are either highly complex (e.g., ridge-in-recess design; Ridgway et al., 1996, Proteins Eng. 9(7):617-621) or use molecules with inadequate stability characteristics (e.g., diantibodies, scFv). In some cases, bispecific antibodies may also have spatial obstruction problems such that both antigens cannot simultaneously bind to each arm of the antibody. Single variable domain antibodies, also known as single domain antibodies or sdAbs, correspond to the variable regions of either the heavy (VH) or light (VL) chains of an antibody. Mouse single domain antibodies are described by Ward et al., 1989, Nature, 341, 544-546. Human single domain antibodies and "camelized" human single domain antibodies have also been described (Holt et al., 2003, Trends in Biotechnology, 21, 484-490). Single domain antibodies are also obtained from camelids (camels and llamas) and cartilaginous fish (carpet sharks and nurse sharks). These organisms have evolved high-affinity single V-like domains (called VHH in camelids and V-NAR in sharks) embedded in the framework region of the Fc-equivalent constant domain as an integral and key component of their immune system (see Holliger & Hudson, for review; 2005, Nature Biotechnology, 23(9):1126-1136).

Therefore, there remains a need to provide additional engineered antibodies, such as fusion proteins, containing an antibody moiety that exhibits one or more desired properties, such as increased functionality (eg, multivalent binding) or improved pharmacokinetics (eg, increased serum half-life). ). It is an object of the present invention to provide a novel engineered antibody format that exhibits increased functionality (eg, increased binding to its antigen, bispecific binding, or multivalent binding) or improved pharmacokinetics (eg, increased serum half-life). In particular, the present invention relates to a new format of bispecific antibodies, in particular, stable and capable of simultaneously binding two antibodies.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an antibody comprising a variable (V) domain and an insert polypeptide, wherein the insert polypeptide is located in the framework region 3 of the V domain.

In some embodiments, the V domain is a VH domain, and the antibody may further comprise a VL domain.

In some embodiments, the V domain is a VL domain, and the antibody may further comprise a VH domain.

In some embodiments, the insert polypeptide is located between amino acid residues 73 and 76 of the VH domain. For example, the insert polypeptide may be in the VH domain: (i) between amino acid residues 73 and 74; (ii) between amino acid residues 74 and 75; or (iii) between amino acid residues 75 and 76.

In some embodiments, one or more amino acid residues between 73 and 76 of the VH domain are replaced with an insert polypeptide.

In some embodiments, the insert polypeptide is located between amino acid residues 67 and 70 of the VL domain. For example, the insert polypeptide may be in the VL domain: (i) between amino acid residues 67 and 68; (ii) between amino acid residues 68 and 69; or (iii) between amino acid residues 69 and 70.

In some embodiments, one or more amino acid residues between residues 67 and 70 of the VL domain are replaced with an insert polypeptide.

In some embodiments, an antibody of the invention further comprises a linker sequence, preferably of at least one, two, three, four, five, six, seven, eight, nine, or ten amino acids, connecting the N- and/or C-terminus of the insert polypeptide to part of the frame region 3.

In some embodiments, an antibody of the invention is a full length antibody or a binding fragment thereof. In some embodiments, the antibody is a full length IgG or a binding fragment thereof. In some embodiments, the antibody is a Fab, Fab', F(ab') ₂ , VHH, or scFv, in particular dsscFv.

In some embodiments, an antibody of the invention is humanized.

In some embodiments, an antibody of the invention comprises a V domain that binds to human serum albumin (HSA). In one embodiment, the antibody contains a VL domain containing a CDR-L1 having the sequence of SEQ ID NO: 3; CDR-L2 having the sequence of SEQ ID NO: 4; CDR-L3 having the sequence of SEQ ID NO: 5. In one embodiment, the antibody contains a VL domain containing SEQ ID NO: 2. In one embodiment, the antibody contains a light (L) chain containing SEQ ID NO: 1. In one embodiment, in an embodiment, the antibody contains a VH domain containing a CDR-H1 having the sequence of SEQ ID NO: 8; CDR-H2 having the sequence of SEQ ID NO: 9; CDR-H3 having the sequence of SEQ ID NO: 10. In one embodiment, the antibody contains a VH domain containing SEQ ID NO: 7. In one embodiment, the antibody contains a heavy (H) chain containing SEQ ID NO: 6.

In some embodiments, the insert polypeptide is a cytokine, preferably wherein the cytokine is selected from IL-10, IL-15, IL-2, G-CSF, GM-CSF, and EPO. In other embodiments, the insert polypeptide is sclerostin. In other embodiments, the insert polypeptide is a second antibody, preferably wherein the second antibody is a scFv, in particular dsscFv, or a domain antibody such as VH or VL or VHH. The VHH may comprise a sequence selected from SEQ ID NOs: 67, 69, and 89. In such embodiments, an antibody may bind an antigen it recognizes via said V domain with a binding affinity that is at least 50%, 60%, 70%, 80% or 90% compared to an antibody without an insert polypeptide.

In some embodiments, the second antibody binds to the same antigen as the V domain. Preferably, the second antibody binds to an epitope other than the V domain. In such embodiments, an antibody can bind an antigen it recognizes with a higher binding affinity than an antibody without a second antibody. In some embodiments, the second antibody binds to an antigen other than the first antibody, i.e. an antibody containing a V domain. In some embodiments, the second antibody binds to IL-17 and the first antibody, i. an antibody containing a V domain binds to HSA. In some embodiments, the second antibody binds to CD3 and the first antibody, i. an antibody containing a V domain binds to CD28. In some embodiments, the second antibody binds to CD28 and the first antibody, i. an antibody containing a V domain binds to CD3.

The present invention also relates to an isolated polynucleotide(s), such as DNA molecule(s), and a cloning or expression vector(s) that encodes an antibody described herein. A host cell containing said polynucleotide(s) or vector(s) is also provided. In addition, the present invention relates to a method for producing an antibody described in the present description, including the cultivation of the specified host cell and isolation of the antibody.

In another aspect, the present invention also provides a pharmaceutical composition comprising an antibody comprising a V domain and an insert polypeptide as described herein, in combination with one or more of a pharmaceutically acceptable excipient, diluent, or carrier. Also provided is said antibody or said pharmaceutical composition for use in therapy. A method of therapy is also provided, comprising administering said antibody or said pharmaceutical composition to an individual in need of therapy. The use of said antibody for the manufacture of a medicinal product is also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: (A) Typical Fab structure highlighting the loop portion of the framework region 3; (B) isolating certain residues in the heavy chain variable framework region 3 region, (C) isolating certain residues in the light chain variable framework region 3 region.

Fig. 2: (A) SPR sensorgram showing CA645/IL-15 _Fwk3 binding to IL-15 receptor chains; (B) Modeled structure showing insertion of IL-15 into framework region 3 of the VH domain of CA645.

Fig. 3: (A) SPR sensorgram demonstrating CA645/IL-2 _Fwk3 binding to the β-chain of the IL-2 receptor with or without the addition of IL-15; (B) Simulated structure showing insertion of IL-2 into framework region 3 of the VH domain of CA645.

Fig. 4: (A) SPR sensorgram showing CA645/G-CSF _Fwk3 binding to HSA and/or G-CSF receptor; (B) Simulated structure showing insertion of G-CSF into framework 3 of the CA645 VH domain.

Fig. 5: (A) SPR sensorgram demonstrating binding of CA645/GM-CSF _Fwk3 (human) to the GM-CSF receptor compared to the CA645/GM-CSF _CH1 construct (human); (B) Simulated structure showing insertion of GM-CSF into framework 3 of the CA645 VH domain.

Fig. 6: SPR sensorgram demonstrating binding of CA645/GM-CSF _Fwk3 (mouse) to the GM-CSF receptor compared to the CA645/GM-CSF _CH1 construct (mouse).

Fig. 7: (A) SPR sensorgram demonstrating CA645/EPO _Fwk3 binding to the EPO receptor with or without the addition of EPO; (B) Simulated structure demonstrating EPO insertion into framework region 3 of the CA645 VH domain.

Fig. 8: (A) SPR sensorgram showing CA645/VHH6 _Fwk3 binding to IL-6, HSA, IL-6:gp80 with or without HSA; (B) Simulated structure showing insertion of VHH6 into framework region 3 of the CA645 VH domain.

Fig. 9: (A) SPR sensorgram showing CA645/VHH15 _Fwk3 binding to HSA and/or IL-6; (B) Simulated structure showing insertion of VHH15 into framework region 3 of the CA645 VH domain.

Figure 10: (A) SPR sensorgram demonstrating binding of CA645/sclerostin _Fwk3 (central or full length) to the extracellular domain of LRP6 with various linker constructs; (B) Simulated structure demonstrating the insertion of the sclerostin core into framework region 3 of the CA645 VH domain.

Fig. 11: SPR sensorgram demonstrating binding of CA645/sclerostin _Fwk3 (central or full length) to the extracellular domain of LRP4 with various linker constructs.

Fig. 12: (A) SPR sensorgram showing the CA645/(IL-10 _Fwk3 ) ₂ construct having different linker sequences binding to the IL-10 receptor; (B) Modeled structure demonstrating IL-10 dimer insertion into framework region 3 of the CA645 VH domain.

Fig. 13: SPR sensorgram demonstrating CA497/IL-15 _Fwk3 binding to IL-2 receptor β chain with or without IL-15 addition.

Fig. 14: SPR sensorgram demonstrating binding of CA240/IL-15 _Fwk3 to the β chain of the IL-2 receptor with or without the addition of IL-15.

Fig. 15: SPR sensorgram showing the dilution series for the binding of HyHEL5/VHH D3L11 _Fwk3 to lysozyme.

Fig. 16: SPR sensorgram showing the dilution series for the HyHEL5/VHH15 _Fwk3 lysozyme binding construct.

Fig. 17: SPR sensorgram showing the dilution series for the CA645/VHH D3L11 _Fwk3 lysozyme binding construct.

Fig. 18: Presentation of a potential binding scheme for HyHEL5/VHH D3L11 _Fwk3 to lysozyme.

Fig. 19: SPR sensorgram demonstrating the CA645/IL-15 _Fwk3 construct having different linker sequences binding to the IL-15 receptor chain.

Fig. 20: DSC curves (solid line: CA645 Fab; dotted line: CA497 dsscFv; inverted triangle: CA645 Fab-Fwk3 CA497 dsscFv (vLvH); triangle: CA645 Fab-Fwk3 CA497 dsscFv (vHvL)).

Fig. 21: Inhibition curves for IL-17A in the absence (solid circle) or presence (open circle) of HSA; (A) CA497 IgG4; (B) CA645 Fab-Fwk3 CA497 dsscFv (vHvL); (C) CA645 Fab-Fwk3 CA497 dsscFv (vLvH).

Fig. 22: SPR analysis demonstrating IL-17A and HSA binding to immobilized CA645 IgG4P construct containing anti-IL-17 dsscFv (vHvL) CA497 embedded in the Fwk3 region.

Fig. 23: DSC curves. (A) Anti-CD28 IgG4P containing anti-CD3 dsscFv in the Fwk3 region (solid line); Anti-CD3 IgG4P containing anti-CD3 dsscFv in the Fwk3 region (dotted line). (B) Anti-CD28 Fab containing anti-CD3 dsscFv in the Fwk3 region (solid line); An anti-CD3 Fab containing anti-CD3 dsscFv in the Fwk3 region (dotted line).

Fig. 24: Analysis of T cell activation in PBMC. Open triangle: anti-CD28 Fab Fwk3-anti-CD3 dsscFv; solid circle: Fab vs. CD3; filled inverted triangle: Fab vs. CD28; solid circle: anti-CD3 Fab Fwk3-anti-CD3 dsscFv; open square: anti-CD28 IgG4P Fwk3-anti-CD3 dsscFv; solid triangle: anti-CD3 IgG4P-Fwk3 anti-CD3 dsscFv; character *: OKT3; open circle: control in the form of cells only. (A) CD4 T cell activation (patient 1), (B) CD4 T cell activation (patient 2) (C) CD8 T cell activation (patient 1) (D) CD8 T cell activation (patient 2).

DETAILED DESCRIPTION

Antibody

The present invention relates to an antibody comprising a variable (V) domain and an insert polypeptide, wherein the insert polypeptide is in the framework region 3 (Fwk3) region of the V domain.

An antibody of the present invention is typically constructed by incorporating an insert polypeptide into the Fwk3 region of the V domain of an antibody. The V domain, for example, may be a VH domain or a VL domain. The antibody in which the insert polypeptide is incorporated may be considered a scaffold antibody and/or, when the insert polypeptide is also an antibody, the antibody in which the insert polypeptide is incorporated may alternatively be referred to as the first antibody, while the insert polypeptide is referred to as second antibody. The terms "framework antibody" and "first antibody" may be used interchangeably in the context of the present invention to refer to an antibody in which an insert polypeptide is incorporated.

In some embodiments, an antibody of the invention is a full length antibody or a binding fragment thereof. In some embodiments, the antibody is a full length IgG or a binding fragment thereof. In some embodiments, the antibody is a full length IgG1. In some embodiments, the antibody is full length IgG4. In some embodiments, the antibody is a full-length IgG4P wherein the serine at position 241 (when numbered according to the Kabat numbering system) has been replaced with a proline. In some embodiments, the antibody is a Fab, Fab', F(ab') ₂ , VHH, or scFv. In some embodiments, the antibody is a disulfide-stabilized scFv or "dsscFv". "Disulfide-stabilized single chain variable fragment" or "dsscFv" as used herein refers to a single chain variable fragment stabilized with a peptide linker between the V _H and V _L variable domains, and also includes an interdomain disulfide bond between V _H and V _L. (See, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012; WO2007109254. In some embodiments, the cross-domain disulfide bond between V _H and V _L is formed between V _H 44 and V _L 100 (numbering according to the Kabat numbering system).

The term "antibody" as used herein generally refers to full length (intact, whole) antibodies, ie. containing elements from two heavy chains and two light chains in the case of an IgG antibody.

The terms "antibody construct", "engineered antibody", "antibody fusion protein" may be used interchangeably in the context of the present invention to refer to an antibody of the invention.

Antibody binding fragments typically contain at least one light chain variable domain (VL) or heavy chain variable domain (VH) and include: single chain antibodies (e.g., full length heavy chain or light chain), Fab, modified Fab, Fab' , modified Fab', F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, single domain antibodies (sdAb, e.g. VH or VL or VHH), scFv, dsscFv, Bis-scFv, diantibodies, tribodies, tribodies, tetraantibodies and epitope-binding fragments of any of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209- 217). Methods for the preparation and production of these antibody binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). For example, antibody binding fragments can be obtained from a whole antibody, especially from a whole monoclonal antibody, using any suitable enzymatic cutting and/or cleavage methods, such as pepsin treatment. Alternatively, antibody starting material can be generated using recombinant DNA techniques involving the manipulation and re-expression of DNA encoding antibody variable and/or constant regions. If desired, standard molecular biology techniques can be used to modify, add or remove amino acids or domains. Any changes to the variable or constant regions are still covered by the terms "variable" and "constant" regions, as used within the scope of the invention. The starting material for the antibody fragment can be from any species, including, for example, mouse, rat, rabbit, hamster, camel, llama, goat, or human. Portions of an antibody fragment may be from more than one species; for example, antibody fragments may be chimeric. In one example, the constant regions are from one species and the variable regions are from another. The starting material for an antibody fragment can also be modified. In another example, the variable region of an antibody fragment is generated using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from naturally occurring antibody variable regions through insertions, deletions, or substitutions into or from the amino acid sequences of naturally occurring antibodies. Specific examples of this type include engineered variable region domains comprising at least one CDR and optionally one or more framework region amino acids from one antibody, and the remainder from the variable region domain from a second antibody. The Fab-Fv format was first described in WO2009/040562, and its disulfide bond-stabilized version of Fab-dsFv was first described in WO2010/035012. Other antibody fragments include the Fab and Fab' fragments described in international patent applications WO2005/003169, WO2005/003170 and WO2005/003171. Polyvalent antibodies may be multispecific, such as bispecific, or may be monospecific (see, for example, WO 92/22583 and WO05/113605). One such example of the latter is Tri-Fab (or TFM) as described in WO92/22583. Another multispecific format is described in WO2015/197772.

A typical Fab' molecule contains a heavy chain and light chain pair, in which the heavy chain contains a VH variable region _, a CH1 constant domain and a natural or modified hinge region, and a light chain contains a VL variable region and a CL constant domain.

The binding domain of an antibody typically contains 6 CDRs, three from the heavy chain and three from the light chain. In such an antibody, the CDRs are in the framework region and together form the variable region. Thus, in one embodiment, the antibody comprises a binding domain specific for an antigen comprising a light chain variable region and a heavy chain variable region. In other embodiments, for example, when the antibody is a single domain antibody, the binding domain may contain only 3 CDRs from a single V domain of either the heavy chain or the light chain. Thus, in one embodiment, the antibody comprises an antigen-specific binding domain comprising one of a light chain variable region (VL domain) or a heavy chain variable region (VH or VHH domain). It will be understood that a V domain that binds an antigen may bind it alone or together with another V domain, eg as a VH/VL complementary pair. Thus, an antibody containing a V domain can bind its antigen together with a complementary V domain (VH or VL). In one embodiment, the V domain is a VH domain, and an antibody of the invention further comprises a VL domain, wherein the VH domain binds its antigen together with the VL domain as a VH/VL pair, i. an antibody of the invention contains a VH domain and binds its antigen together with a complementary VL domain. In one embodiment, the V domain is a VL domain, and an antibody of the invention further comprises a VH domain, wherein the VL binds its antigen together with the VH domain as a VH/VL pair, i. an antibody of the invention contains a VL domain and binds its antigen together with a complementary VH domain.

The binding domain of an antibody of the invention generally refers to the VH, VL, or VH/VL pair that binds the antigen.

Antibodies typically contain one or more binding domains. For example, an unmodified Fab fragment contains one binding domain formed by a VH/VL pair and a whole IgG contains two binding domains each formed by a VH/VL pair, where the two binding domains are identical (monospecific antibody) or different (bispecific or bivalent). antibody). In one example, an antibody of the invention may be a Fab fragment containing a VH domain and an insert polypeptide in the Fwk3 region of the VH domain, and further comprising a VL domain that is complementarily paired with the VH domain. In one example, the VL domain may be unmodified (i.e., an antibody of the invention is a Fab fragment containing a single insert polypeptide in the Fwk3 region of the VH domain) or may contain an insert polypeptide in its Fwk3 region (i.e., an antibody of the invention is a Fab fragment containing two insert polypeptides identical, similar or different, one of which is inserted into the Fwk3 region of the VH domain and the other is inserted into the Fwk3 region of the VL domain). In one example, an antibody of the invention may be a Fab fragment containing a VL domain and an insert polypeptide in the Fwk3 region of the VL domain, and further comprising a VH domain that forms a complementary pair with the VL domain. In one example, the VH domain may be unmodified (i.e., an antibody of the invention is a Fab fragment containing a single insert polypeptide in the Fwk3 region of the VL domain) or may contain an insert polypeptide in its Fwk3 region (i.e., an antibody of the invention is a Fab fragment containing two identical, similar or different insert polypeptides, one of which is inserted in the Fwk3 region of the VL domain and the other is inserted in the Fwk3 region of the VH domain). In another example, an antibody of the invention is a complete IgG containing a VH domain and an insert polypeptide in the Fwk3 region of the VH domain, and additionally containing a VL domain that forms a complementary pair with the VH domain. In one example, the VL domain may be unmodified (i.e., an antibody of the invention is an IgG containing two insert polypeptides, each inserted into one of two IgG VH domains) or may contain an insert polypeptide in its Fwk3 region ( ie, an antibody of the invention is an IgG containing four identical, similar or different insert polypeptides, each inserted into the Fwk3 region of each of two VH domains and two VL domains). In another example, an antibody of the invention is a complete IgG containing a VL domain and an insert polypeptide in the Fwk3 region of the VL domain, and additionally containing a VH domain that forms a complementary pair with the VL. The VH domain may be unmodified (i.e., an antibody of the invention is an IgG containing two insert polypeptides each inserted into one of two IgG VL domains) or may contain an insert polypeptide in its Fwk3 region (i.e. The antibody of the invention is an IgG containing four identical, similar or different insert polypeptides, each inserted into the Fwk3 region of each of two VL domains and two VH domains).

Residues in the variable domains of antibodies are usually numbered according to the system developed by Kabat et al. This system is listed in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA. In the present description, this numbering system is used except where otherwise indicated.

Residue designations according to Kabat do not always directly correspond to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids relative to the strict Kabat numbering, corresponding to a truncation or insertion into a structural component, either a framework region or a complementarity determining region (CDR) of the main structure of the variable domain. The correct Kabat residue numbering can be determined for a given antibody by aligning the homology residues in the antibody sequence with the "standard" Kabat numbered sequence by methods known in the art.

The heavy chain variable domain CDRs are at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. The light chain variable domain CDRs are at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. In addition to the CDR loops, there is a fourth loop between CDR-2 and CDR-3, formed by frame region 3 (Fwk3). In FIG. 1A shows a frame loop 3 in the context of a Fab having light and heavy chains. In FIG. 1B and 1C also show representative remnants from these loops. The Kabat numbering system defines framework 3 as heavy chain positions 66-94 and light chain positions 57-88.

Thus, in one embodiment, an antibody of the present invention comprises a variable (V) domain and an insert polypeptide, wherein the insert polypeptide is at positions 66-94 when the V domain is a VH domain, or at positions 57-88, when the V-domain is a VL-domain, according to the Kabat numbering system.

In some embodiments, the insert polypeptide is located between amino acid residues 73 and 76 of the VH domain, according to the Kabat numbering system. Preferably, the insert polypeptide may be between amino acid residues 73 and 74, 74 and 75, or 75 and 76 of the VH domain, according to the Kabat numbering system. In some embodiments, the insert polypeptide is between amino acid residues 76 and 77, or 77 and 78 of the VH domain, according to the Kabat numbering system. In some embodiments, one or more amino acid residues of the VH domain are replaced with an insert polypeptide.

In some embodiments, the insert polypeptide is located between amino acid residues 67 and 70 of the VL domain according to the Kabat numbering system. Preferably, the insert polypeptide may be between amino acid residues 67 and 68, 68 and 69, or 69 and 70 of the VL domain according to the Kabat numbering system. In some embodiments, one or more amino acid residues of the VL domain are replaced with an insert polypeptide.

An antibody of the present invention may further comprise constant region domains. The domains of the constant region of the antibody molecule of the present invention, if present, can be selected based on the intended function of the antibody molecule and, in particular, the effector functions that may be required. For example, constant region domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, human IgG constant region domains, especially of the IgG1 and IgG3 isotypes, can be used when antibody effector functions are required. Alternatively, IgG2 and IgG4 isotypes can be used when antibody effector functions are not required. It will be appreciated that sequence variants of these constant region domains may also be used. For example, IgG4 molecules can be used in which serine at position 241 is replaced by proline, as described in Angal et al. Molecular Immunology, 1993, 30(1), 105-108. It will also be appreciated by one of ordinary skill in the art that antibodies may undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include variation in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

Antibodies for use in the present invention may be fully human antibodies, humanized antibodies, or chimeric antibodies.

In one embodiment, the antibody of the present invention is not a mouse antibody. The term "mouse antibody" refers to an antibody having heavy and light chains derived exclusively from mouse B cells. Thus, this antibody consists of mouse amino acid sequences, regardless of the origin of the cells that provide their production.

In one embodiment, the antibody of the present invention is a chimeric antibody. The term "chimeric antibody" refers to an antibody or antigen-binding fragment thereof, where each light chain and/or heavy chain sequence of which it consists, contains or consists of a hybrid sequence derived from at least two different animals, especially mammals, in particular , to an antibody in which a rabbit, mouse, rat, or cynomolgus monkey variable region is combined with a human constant region.

In one embodiment, the antibody of the present invention is a humanized antibody. Humanized antibodies are antibody molecules from a non-human species having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, for example, US 5,585,089). In particular, the term "humanized" refers to an antibody or antigen-binding fragment thereof, where the heavy and/or light chain contains one or more CDRs from a non-human antibody (such as a rabbit, mouse, rat, cynomolgus or llama monoclonal antibody) transplanted into the framework region of the heavy and/or light chain variable region of an acceptor antibody, such as a human antibody. Instead of transferring an entire CDR, only one or more of the specificity-determining residues from any of the CDRs described herein above can be transferred into the framework region of a human antibody (see, for example, Kashmiri et al., 2005, Methods, 36, 25- 34). Individual framework region residues of a humanized antibody can be replaced with corresponding residues (so-called donor residues) from a non-human antibody (e.g., the antibody from which the CDRs or specificity-determining residues are derived), e.g., to restore or increase the specificity or affinity of the antibody.

Fully human antibodies are antibodies in which the variable region(s) and constant region(s) (when present) of the heavy and/or light chains are fully human in origin or substantially identical to sequences of human origin, not necessarily from the same antibodies. Examples of fully human antibodies may include antibodies obtained, for example, by phage display methods, and antibodies produced by mice in which the mouse immunoglobulin variable and/or constant region genes are replaced with human analogues, for example, as generally described in EP0546073 B1, US 5545806 , US 5569825, US 5625126, US 5633425, US 5661016, US 5770429, EP 0438474 B1 and EP0463151 B1.

Antibodies containing a V domain and an insert polypeptide as described herein can be further modified, such as chemically conjugated with other compounds, which may affect, for example, their pharmacokinetic properties or antigen binding.

In one example, the antibodies of the present invention are linked to poly(ethylene glycol) (PEG) moieties. PEG-linked antibodies may, for example, exhibit further increased serum half-life. In one specific example, an antibody is an antibody fragment and the PEG molecules can be linked through any available amino acid side chain or terminal amino acid functional group found in the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may be naturally occurring in the antibody fragment, or may be inserted into the fragment using recombinant DNA techniques (see, for example, US 5219996; US 5667425; WO98/25971, WO2008/038024). In one example, an antibody of the present invention is a modified Fab fragment, where the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which an effector molecule can be linked. Multiple sites can be used to attach two or more PEG molecules.

Suitably, the PEG molecules may be covalently linked through the thiol group of at least one cysteine residue present in the antibody fragment. Each polymer molecule associated with the modified antibody fragment may be covalently linked to the sulfur atom of a cysteine residue found in the antibody fragment. The covalent bond is typically a disulfide bond or, in particular, a sulfur-carbon bond. When a thiol group is used as the point of attachment, suitably activated effector molecules can be used, for example thiol-selective derivatives such as maleimides and cysteine derivatives. An activated polymer can be used in the preparation of polymer-modified antibody fragments as described above. The activated polymer may be any polymer containing a thiol-reactive group such as an α-halocarboxylic acid or an ester, eg iodoacetamide, an imide, eg maleimide, vinyl sulfone or disulfide. Such starting materials may be obtained from commercial sources (eg, from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or may be obtained from commercially available starting materials using conventional chemical techniques. Specific PEG molecules include methoxy-PEG-amine 20K (obtained from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (obtained from Nektar, formerly Shearwater).

Preferably, an antibody of the present invention has a binding affinity (as measured by its dissociation constant K _D ) for the antigen it recognizes of 10 ^-5 M or less, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, or 10 ^-11 M or less. Affinity can be measured by known methods such as surface plasmon resonance methods including Biacore™. Affinity can be measured at room temperature, 25°C or 37°C. Affinity can be measured at physiological pH values, ie. at approximately pH 7.4.

It will be understood that the affinity of the antibodies provided by the present invention can be changed using any suitable method known in this field. The present invention thus also relates to variants of the antibody molecules of the present invention which have an increased affinity for albumin. Such variants can be generated through a variety of affinity maturation protocols, including CDR mutation (Yang et al., J. Mol. Biol, 254. 392-403, 1995), strand shuffling (Marks et al., Bio/Technology, 10, 779- 783, 1992), use of E. coli mutator strains (Low et al., J. Mol. Biol., 260. 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. BiotechnoL, 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol, 256. 77-88, 1996) and sex PCR (Crameri et al.. Nature, 391. 288-291, 1998).

Human serum albumin binding antibody

In one embodiment, an antibody of the invention contains a V domain that binds to human serum albumin (HSA). In such an embodiment, the antibody may be useful for extending the serum half-life of the insert polypeptide. It will be appreciated that the V domain that binds albumin can also bind alone or in conjunction with another V domain, eg as a VH/VL complementary pair. Thus, in some embodiments, an antibody containing a V domain binds albumin together with a complementary VH or VL domain. In one embodiment, the V domain is a VH domain, and the antibody of the invention further comprises a VL domain, wherein the VH binds albumin together with the VL domain as a VH/VL pair, i. an antibody of the invention contains a VH domain and binds to albumin in conjunction with a complementary VL. In one embodiment, the V domain is a VL domain and the antibody of the invention further comprises a VH domain, wherein the VL binds albumin together with the VH domain as a VH/VL pair, i. an antibody of the invention contains a VL domain and binds to albumin in conjunction with a complementary VH. When an antibody of the invention binds albumin, VH/VL is generally referred to as an "albumin binding domain". In this regard, the albumin-binding domain of an antibody of the invention refers to a VH, VL, or VH/VL pair that binds to albumin.

In one embodiment, an antibody of the present invention binds cynomolgus monkey serum albumin, mouse serum albumin, and/or rat serum albumin.

In one embodiment, an antibody of the present invention containing a V domain that binds to human serum albumin is humanized. In one embodiment, an albumin-binding domain-containing antibody of the present invention is a humanized antibody, wherein the heavy and/or light chain comprises one or more CDRs from a rabbit, mouse, or rat monoclonal antibody grafted into the heavy and/or light variable region framework. human antibody chains. In one embodiment, an albumin-binding domain-containing antibody of the present invention is a humanized antibody, wherein the heavy and/or light chain comprises one or more CDRs from rabbit antibodies transplanted into the heavy and/or light chain variable region framework of a human antibody, and wherein the heavy and/or light chain variable region framework contains at least one amino acid substitution, such as an amino acid that is a donor residue.

In one embodiment, an antibody of the present invention containing a V domain that binds to human serum albumin contains a light and/or heavy chain sequence as shown below (variable domain underlined; CDRs in bold and italics). In one embodiment, an antibody of the present invention containing a V domain that binds to human serum albumin contains a light and/or heavy chain sequence selected from the sequences below. In one embodiment, an antibody of the present invention comprising a V domain that binds to human serum albumin comprises a light and/or heavy chain variable domain sequence selected from the following sequences. In one embodiment, an antibody of the present invention containing a V domain that binds to human serum albumin contains at least one of the sequences of CDR-L1, CDR-L2 and CDR-L3 and/or at least one of the sequences of CDR-L1, CDR-L2 and CDR-L3 H1, CDR-H2 and CDR-H3 selected from the following sequences. In one embodiment, an antibody of the present invention containing a V domain that binds to human serum albumin contains a light and/or heavy chain sequence; and/or a light and/or heavy chain variable domain sequence; and/or at least one of the sequences CDR-L1, CDR-L2 and CDR-L3; and/or at least one of the CDR-H1, CDR-H2 and CDR-H3 sequences selected from the following sequences.

Light chain CA645 (gL5)

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1)

VL domain (gL5):

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIK (SEQ ID NO: 2)

CDR-L1: QSSPSVWSNFLS (SEQ ID NO: 3)

CDR-L2: EASKLTS (SEQ ID NO: 4)

CDR-L3: GGGYSSISDTT (SEQ ID NO: 5)

Heavy chain CA645 (gH5)

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 6)

VH domain (gH5): EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS (SEQ ID NO: 7)

CDR-H1: GIDLSNYAIN (SEQ ID NO: 8)

CDR-H2: IIWASGTTFYATWAKG (SEQ ID NO: 9)

CDR-H3: TVPGYSTAPYFDL (SEQ ID NO: 10)

In some embodiments, an antibody of the present invention comprises human serum albumin binding VL and VH domain variants as described above (SEQ ID NOs: 2 and 7, respectively) that contain an additional residue such that between VL and VH domains can form a disulfide bond. Additional cysteine-containing variants may have the following sequences (where additional cysteine residues are underlined):

VL domain CA645-Cys (gL5):

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIK (SEQ ID NO: 102)

VH domain of CA645-Cys (gH5):

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS (SEQ ID NO: 103)

Additional examples of VL and VH domains that bind human serum albumin and that can be used in an antibody of the invention include SEQ ID NOS: 105, 106, 107, 108, and 109, as described below (CDRs in bold and italics).

VH domain of CA645 (gH1):

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDSTTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS (SEQ ID NO: 105)

VH domain of CA645 (gH37):

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTAYATWAKGRFTI SRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS (SEQ ID NO: 106)

VH domain of CA645 (gH47):

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDAVYYCARTVPGYSAAPYFDLWGQGTLVTVSS (SEQ ID NO: 107)

VL domain of CA645 (gL1):

DIVMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTS GVPSRFKGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIK (SEQ ID NO: 108)

VL domain of CA645 (gL4):

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC GGGYSSISD TTFGGGTKVEIK

(SEQ ID NO: 109)

In some embodiments, an antibody of the present invention comprises human serum albumin binding VL and VH domain variants as described above (SEQ ID NO: 105-SEQ ID NO: 109) that contain an additional cysteine residue such that between VL- and VH domains can form a disulfide bond. Additional cysteine-containing variants may have the following sequence (where the additional cysteine residue is underlined):

VL CA645-Cys (gL4):

diqmtqspssvsasvgdrvtitcqsspsvwsnflswyqqkpgkapklliyeaskltsgvpsrfsgsgsgtdftltisslqpedfatyycgggyssisdttfgCgtkveikRT(SEQ ID NO: 110)

In some embodiments, the framework region of the VH albumin binding domain is human (e.g., VH3, such as VH3 1-3 3-23), and contains, for example, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as amino acids, which are donor residues. In such embodiments, the VH may have the sequence shown in SEQ ID NO: 7, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or a variant of any of these having at least least 95, 96, 97, 98 or 99% similarity or identity.

In some embodiments, the framework region of the VL albumin-binding domain is human (e.g., Vκ1, such as 2-1-(1)L5), and contains, for example, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as amino acids , which are donor residues. In such embodiments, the VL may have the sequence shown in SEQ ID NO: 2, SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or a variant of any of these having at least least 95, 96, 97, 98 or 99% similarity or identity.

In some embodiments, the albumin binding domain comprises VH and VL sequences selected from combinations of SEQ ID NOs: 7 and 2, 7 and 102, 7 and 108, 7 and 109, 7 and 110, 103 and 2, 103 and 102, 103 and 108 , 103 and 109, 103 and 110, 105 and 2, 105 and 102, 105 and 108, 105 and 109, 105 and 110, 106 and 2, 106 and 102, 106 and 108, 106 and 109, 106 and 110, 107 and 2, 107 and 102, 107 and 108, 107 and 109, 107 and 110, or a variant or variants of any of them having at least 95%, 96%, 97%, 98% or 99% similarity or identity.

In some embodiments, the VL and VH sequences of the albumin binding domain are SEQ ID NO:102 and SEQ ID NO:103, respectively. In some embodiments, the VL and VH sequences of the albumin binding domain are SEQ ID NO:2 and SEQ ID NO:7, respectively.

In one embodiment, the V-domain-containing antibody that binds human serum albumin is a Fab or scFv. The VL and VH domains of SEQ ID NOs: 2 and 7 are preferred for the Fab format. In one embodiment, an antibody containing a V domain that binds human serum albumin is a scFv that binds albumin together with a complementary V domain (VL or VH). In such an embodiment, the scFv may comprise any of the VH domains and any of the VL domains as described above linked in a VH-VL or VL-VH orientation via a (G4S) ₄ linker (SEQ ID NO:16). The VL and VH domains of SEQ ID NOs: 2 and 7 are preferred for the scFv format. The VL domain of SEQ ID NO: 2 may contain an additional two RT amino acids at the C-terminus. In one embodiment, the V domain-containing antibody that binds human serum albumin is a scFv that contains the VL and VH domains of SEQ ID NOs: 2 and 7 separated by a (G4S) ₄ linker. In one example, the scFv has a VH-VL orientation. In another example, the scFv is VL-VH oriented. In one embodiment, the V-domain-containing antibody that binds human serum albumin is a scFv that contains or has SEQ ID NO: 143 as shown below. In one specific embodiment, the V-domain-containing antibody that binds human serum albumin is dsscFv. In one embodiment, an antibody containing a V domain that binds human serum albumin is a dsscFv that binds albumin together with a complementary V domain (VL or VH). In such an embodiment, the dsscFv may contain any VH domains and any VL domains as described above, containing, if not already present, an additional cysteine residue such that a disulfide bond can be formed between the VL and VH domains, where VL is and the VH domains are linked in a VH-VL or VL-VH orientation via a (G4S) ₄ linker. The VL and VH domains of SEQ ID NOs: 102 and 103 are preferred for the dsscFv format. The VL domain of SEQ ID NO: 102 may contain an additional two RT amino acids at the C-terminus. In one embodiment, the antibody containing the V domain that binds human serum albumin is dsscFv, which contains the VL and VH domains of SEQ ID NO: 102 and 103 separated by a linker (G4S) ₄ (SEQ ID NO:16) . In one example, dsscFv has a VH-VL orientation. In another example, dsscFv has a VL-VH orientation. In one embodiment, the V-domain-containing antibody that binds human serum albumin is a dsscFv that contains or has SEQ ID NO: 144 as shown below.

CA645 scFv (VH-VL):

evqllesggglvqpggslrlscavsgidlsnyainwvrqapgkGlewigiiwasgttfyatwakgrftisrdnskntvylqmnslraedtavyycartvpgystapyfdlwgqgtlvtvssggggsggggsggggsggggsdiqmtqspssvsasvgdrvtitcqsspsvwsnflswyqqk pgkapklliyeaskltsgvpsrfsgsgsgtdftltisslqpedfatyycgggyssisdttfgQgtkveik (SEQ ID NO: 143)

dsscFv (VH-VL) CA645, where additional cysteine residues are underlined:

evqllesggglvqpggslrlscavsgidlsnyainwvrqapgkclewigiiwasgttfyatwakgrftisrdnskntvylqmnslraedtavyycartvpgystapyfdlwgqgtlvtvssggggsggggsggggsggggsdiqmtqspssvsasvgdrvtitcqsspsvwsnflswyqqkp gkapklliyeaskltsgvpsrfsgsgsgtdftltisslqpedfatyycgggyssisdttfgcgtkveik (SEQ ID NO: 144)

In another embodiment, the V-domain-containing antibody that binds human serum albumin is a full-length IgG. The VL and VH domains of SEQ ID NOs: 2 and 7 are preferred for the IgG format. In one embodiment, the V-domain-containing antibody that binds human serum albumin is IgG1. In one embodiment, the V-domain-containing antibody that binds human serum albumin is IgG4. In one embodiment, the V-domain-containing antibody that binds human serum albumin is IgG4P.

It will be understood that one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions, and/or deletions can be made to CDRs or other sequences (e.g., variable domains) contemplated by the present invention without significantly altering the ability of the antibody to bind with an antigen it recognizes (for example, HSA). The effect of any amino acid substitutions, insertions and/or deletions can be easily tested by a person skilled in the art. For example, one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions, and/or deletions can be introduced into a framework used in an antibody or fragment of the present invention, where the binding affinity for the antigen they recognize is maintained or increased. . In some embodiments, the amino acid at position 72 of framework region 3 of the VH domain is a wild-type amino acid naturally present at position 72 of said VH domain.

In this regard, the present invention also relates to antibodies that contain sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % similar or identical to the sequence given in the present description. "Identity" as used herein refers to any particular position in aligned sequences where an amino acid residue is identical between sequences. "Similarity" as used herein indicates that at any particular position in the aligned sequences, the amino acid residue is an amino acid residue of a similar type between the sequences. For example, leucine can be replaced by isoleucine or valine. Other amino acids that are often substituted for other amino acids include, but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

- lysine, arginine and histidine (amino acids having basic side chains);

- aspartate and glutamate (amino acids having acidic side chains);

- asparagine and glutamine (amino acids having amide side chains); And

- cysteine and methionine (amino acids having sulfur-containing side chains).

Degrees of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993 ; Computer Analysis of Sequences Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, the BLAST™ software available from NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215:403- 410 Gish, W. & States, D. J. 1993, Nature Genet 3:266-272 Madden, T. L. et al., 1996, Meth Enzymol 266:131-141 Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649-656).

An antibody of the present invention can bind to albumin with a binding affinity sufficient to extend the half-life of an insert polypeptide, such as a therapeutic protein or second antibody, in vivo. In one embodiment, the insert polypeptide binds to HSA. In such an embodiment, the insert polypeptide can bind to albumin with a binding affinity sufficient to prolong the half-life of the scaffold antibody. It has been described that a K _D for albumin of less than or equal to 2.5 μM will prolong in vivo half-life (Nguyen, A. et al (2006) Protein Engineering, Design & Selection, 19(7), 291-297). In one example, an antibody of the present invention can bind to albumin with a high binding affinity that is nanomolar or micromolar, eg, with a K _D equal to or less than 3 nM. Affinity can be measured using any suitable method known in the art, including surface plasmon resonance using natural or recombinant serum albumin.

In some embodiments, the insert polypeptide is an albumin binding peptide. Examples of albumin binding peptides are described in WO2007/106120 and include:

SEQID NO: SUBSEQUENCE 111 DLCLRDWGCLW 112 DICLPRWGCLW 113 MEDICLPRWGCLWGD 114 QRLMEDICLPRWGCLWEDDE 115 QGLIGDICLPRWGCLWGRSV 116 QGLIGDICLPRWGCLWGRSVK 117 EDICLPRWGCLWEDD 118 RLMEDICLPRWGCLWEDD 119 MEDICLPRWGCLWEDD 120 MEDICLPRWGCLWED 121 RLMEDICLARWGCLWEDD 122 EVRSFCTRWPAEKSCKPLRG 123 RAPESFVCYWETICFERSEQ 124 EMCYFPGICWM

In one embodiment, the insert polypeptide is a second antibody that binds to HSA. In such an embodiment, the insert polypeptide may have the features described above for an antibody containing a V domain, ie. framework antibody. In some embodiments, the insert polypeptide may comprise at least one of the CDRs, at least one of the VL or VH domains, or at least one of the light or heavy chains that bind albumin as described above. In one embodiment, the insert polypeptide comprises at least one of CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and/or one of CDR-H1, CDR-H2 and CDR-H3 with the sequence SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

Insert polypeptide

In accordance with the present invention, the present invention relates to antibodies containing an insert polypeptide in the frame region 3 of the V domain. The V domain may be a VL or VH domain. In one example, an antibody of the invention may contain an insert polypeptide in both VL and VH framework region 3, i. e. an antibody of the invention may comprise two insert polypeptides, wherein one insert polypeptide is inserted into a 3 VL framework region and one insert polypeptide is inserted into a 3 VH framework region, and wherein the insert polypeptides are identical, similar, or different. In one embodiment, the insert polypeptide is heterologous to the antibody. As used herein, "heterologous" generally means from an environment that is genotypically different from the rest of the environment to which it is being compared. In this context, this means that the inserted polypeptide is not normally present in the native sequence of the antibody. In one embodiment, the insert polypeptide is a full length protein. In one embodiment, the insert polypeptide is a fragment of a full length protein. In one embodiment, the insert polypeptide is a functional fragment of a full length protein. In one embodiment, the insert polypeptide is functional when expressed on its own. In one embodiment, the insert polypeptide is functional and/or retains functionality when present in the framework region 3 region of the V domain of an antibody of the invention. Thus, in one embodiment, the present invention provides an antibody comprising a variable (V) domain and an insert polypeptide, wherein the insert polypeptide is located in the framework region 3 (Fwk3) region of the V domain, and wherein the insert polypeptide is functional and/or retains functionality when it is present in the framework region 3 in the V domain of an antibody of the invention.

In one embodiment, the insert polypeptide contains at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 200 amino acids. In one embodiment, the insert polypeptide contains less than 250, 300, or 350 amino acids. In one embodiment, the insert polypeptide contains less than 250 amino acids. In one embodiment, the insert polypeptide contains at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 200 amino acids and less than 250 amino acids. In one embodiment, the insert polypeptide contains 50 to 250, preferably 100 to 250, more preferably 150 to 250 amino acids.

In one embodiment, the insert polypeptide may be a therapeutic or diagnostic polypeptide. Suitable therapeutic polypeptides may include, for example, receptor agonists or antagonists, enzyme inhibitors, metal chelators, antivirals, antifungals, cardiovascular drugs, and chemotherapy drugs. In some embodiments, the insert polypeptide is a cytokine. The cytokine may be selected from, for example, IL-10, IL-15, IL-2, G-CSF, GM-CSF, EPO. In other embodiments, the insert polypeptide is sclerostin. In some embodiments, the insert polypeptide contains more than one polypeptide sequence, eg, two copies of the same polypeptide, optionally linked. Advantageously, in such an embodiment, the insert polypeptide may form a multimer, in particular a functional multimer. In some embodiments, the insert polypeptide contains two, three, or four polypeptide sequences and is capable of forming a dimer, trimer, or tetramer of the polypeptide. In some embodiments, the insert polypeptide forms a homodimer, homotrimer, or homotetramer. In some embodiments, the insert polypeptide contains at least two cytokines. In one embodiment, the insert polypeptide contains two human IL-10 sequences, optionally separated by a peptide linker, for example, (G4S)3 (SEQ ID NO: 15), i.e. two human IL-10 sequences are inserted into the Fwk3 region of the V domain of an antibody of the invention. In such an embodiment, two human IL-10 sequences form an IL-10 homodimer included in an antibody of the invention.

In some embodiments, the insert polypeptide is a chemokine. The chemokine can be selected from, for example, CCL (C-C motif chemokine ligands), CXCL (CXC motif chemokine ligands). In some embodiments, the insert polypeptide is a hormone. The hormone may be selected from, for example, insulin or leptin. In some embodiments, the insert polypeptide is a growth factor.

Surprisingly, the present inventors have found that insertion of a polypeptide into an antibody in the V-domain 3 framework loop region does not significantly interfere with the functionality of either the antibody or the insert polypeptide. In particular, the examples herein show that specific and recognition-driven binding to the respective binding partners of both the antibody and the insert polypeptide is observed for several of the fusion protein constructs tested.

In some embodiments, an antibody of the invention binds to an antigen it recognizes via said V domain with a binding affinity that is at least 50%, 60%, 70%, 80%, or 90% compared to an antibody without an insert polypeptide. It will be understood that a V domain that binds an antigen may bind it alone or together with another V domain depending on the format of the antibody, eg as a VH/VL complementary pair. Thus, an antibody containing a V domain can bind its antigen together with a complementary V domain (VH or VL). In some embodiments, an antibody of the invention binds an antigen it recognizes with a binding affinity that is at least 50%, 60%, 70%, 80%, or 90% compared to an antibody without an insert polypeptide. In some embodiments, an antibody of the invention contains a VH domain and binds its antigen together with a complementary VL with a binding affinity that is at least 50%, 60%, 70%, 80%, or 90% compared to an antibody without an insert polypeptide. In some embodiments, an antibody of the invention contains a VL domain and binds its antigen together with a complementary VH domain with a binding affinity that is at least 50%, 60%, 70%, 80%, or 90% compared to an antibody without a polypeptide inserts. In some embodiments, the insert polypeptide antibody portion binds a binding partner it recognizes with a binding affinity that is at least 50%, 60%, 70%, 80%, or 90% relative to the free polypeptide when not bound to antibody.

Thus, in one embodiment, an antibody of the invention comprises a variable (V) domain and an insert polypeptide, wherein the insert polypeptide is in the framework region 3 (Fwk3) region of the V domain, and wherein both the V domain containing antibody and the polypeptide -insert are functional and/or retain functionality when the insert polypeptide is present. In such embodiments, an antibody of the invention has the functionality of both a scaffold antibody and an insert polypeptide. Advantageously, an antibody of the invention may have both the functionality of both a scaffold antibody and an insert polypeptide.

An antibody containing a variable (V) domain is considered to be functional and/or functional when the insert polypeptide is present in the framework region 3 region of the V domain, where the antibody of the invention exhibits at least 50%, 60%, 70%, 80% or 90% of at least one of the functions of an antibody containing a variable (V) domain without an insert polypeptide. An insert polypeptide is considered functional and/or functional when it is present in a V domain framework 3 where the insert polypeptide exhibits at least 50%, 60%, 70%, 80%, or 90% of at least one of the functions the insert polypeptide when it is self-expressed, i.e. compared to the free polypeptide when it is not bound to an antibody.

The "functional" or "functionality" of an antibody and/or insert polypeptide refers to at least one biological activity of said antibody and/or insert polypeptide, including, for example, target binding (e.g., antigen binding), binding affinity, cross-reactivity, neutralization , serum half-life, activation of cell surface proteins, eg T-cell surface receptors. Methods for determining functionality, eg binding affinity, neutralization and activation of target cells are well known in the art and include, for example, the methods described in the examples described herein. For example, standard assay methods such as Scatchard assay or surface plasmon resonance (eg, using BIACORE®) can be used to determine target binding (eg, antigen binding) and binding affinity.

Surprisingly, the V domain (VL or VH domain) of an antibody of the invention can still form a functional binding domain with its corresponding or complementary V domain (VH or VL) when present.

In some embodiments, the insert polypeptide may be a second antibody or a binding fragment thereof. In such embodiments, the insert polypeptide may have any of the antibody features as described above. In particular, in some embodiments, the insert polypeptide is scFv, and dsscFv, a single domain antibody such as VH or VL or VHH. In some embodiments, the insert polypeptide may be a fully human, humanized, or chimeric antibody. An insert polypeptide may contain one or more CDRs, or may be composed of one or more CDRs. For example, the insert polypeptide may comprise or consist of at least one of CDR-H1, CDR-H2, CDR-H3 and/or CDR-L1, CDR-L2, CDR-L3, optionally linked.

Those skilled in the art will appreciate that it can function as a divalent antibody or a bispecific antibody. Thus, in one aspect, the invention relates to a new bivalent or bispecific format capable of simultaneously binding two different epitopes. In some aspects, epitopes are different epitopes present on the same antigen. In another aspect, the invention relates to a new format of bispecific antibodies capable of simultaneously binding two different antigens.

In one embodiment, the antigen of interest bound by the first or second antibody, or antibody fragment, may be a cell-associated protein, e.g., a cell surface protein on cells such as bacterial cells, yeast cells, T cells, endothelial cells, or tumor cells. cells, or it may be a soluble protein. The antigens of interest can also be any protein of medical importance, such as proteins that are elevated in the course of a disease or infection, such as receptors and/or their respective ligands. Specific examples of cell surface proteins include adhesion molecules, e.g., integrins such as β1-integrins, e.g. , CDllb, GDIS, CD19, CD20, CD23, CD25, CD28, CD33, CD38, CD40, CD45, CDW52, CD69, CD 134 (OX40), ICOS, BCMP7, CD 137, CD27L, CDCP1, DPCR1, DPCR1, dudulin 2 , FLJ20584 FLJ40787 HEK2 KIAA0634 KIAA0659 KIAA1246 KIAA1455 FTBP2 FTK , DTD, carcinoembryonic antigen (CEA ), human human milk fat globulin (HMFG1 and 2), MHC class I and MHC class II antigens, and VEGF, and, when appropriate, their receptors. Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-16 or IL-17, viral antigens, for example, respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins such as IgE, interferons such as interferon α, interferon β or interferon γ, tumor necrosis factor α, tumor necrosis factor β, colony stimulating factors such as G-CSF or GM- CSF, and platelet growth factors such as PDGF-α and PDGF-β and, when appropriate, their receptors. Other antigens include bacterial cell surface antigens, bacterial toxins, viruses such as influenza virus, EBV, Hep A, B and C, bioterrorism agents, radionuclides and heavy metals, and venoms and toxins from snakes and spiders.

In some embodiments, neither the framework antibody nor the insert polypeptide binds (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP) hapten. In some embodiments, the antibody is different from the mouse BI-8 antibody.

In one embodiment, the second antibody binds to an antigen other than the V domain of the antibody.

In other embodiments, the second antibody binds to the same antigen as the V domain of the antibody. Preferably, the third antibody binds to an epitope other than that of the V domain of the antibody. In such embodiments, the antibody can bind the antigen it recognizes with a binding affinity that is greater than the binding affinity of the antibody without the second antibody.

In one embodiment, the insert polypeptide is a second antibody that binds IL-17, in particular IL-17A. In one embodiment, the insert polypeptide is a second antibody that binds IL-17AA, IL-17AF, and IL-17FF. In one embodiment, the second antibody that binds IL-17 is scFv. In one embodiment, the second antibody that binds IL-17 is dsscFv. In one embodiment, the antibody of the present invention is a Fab fragment that binds to both HSA and IL-17, in particular, the antibody containing the V domain binds to HSA, and the insert polypeptide is a dsscFv that binds IL-17 i.e. the framework antibody is a Fab fragment that binds HSA and the second antibody inserted into the Fwk3 region of the V domain of the framework antibody is dsscFv that binds IL-17. In another embodiment, the antibody of the invention is a whole IgG that binds to both HSA and IL-17, in particular the V-domain containing antibody binds to HSA and the insert polypeptide is a dsscFv that binds IL -17, i.e. the framework antibody is a full IgG that binds HSA, and the second antibody inserted into the Fwk3 region of the V domain of the framework antibody is dsscFv that binds IL-17. In one embodiment, an antibody of the invention is a Fab fragment that contains or has the heavy and light chains of SEQ ID NO: 129 (heavy chain Fab CA645 with dsscFv CA497 (vHvL) embedded in framework 3) and SEQ ID NO: 1 , respectively, or a variant or variants of any of them having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity, provided that they retain their functionality, for example, binding to HSA and/or IL-17. In one embodiment, an antibody of the invention is a Fab fragment that contains or has the heavy and light chains of SEQ ID NO: 130 (heavy chain Fab CA645 with dsscFv CA497 (vLvH) embedded in framework 3) and SEQ ID NO: 1 , respectively, or a variant or variants of any of them having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity, provided that they retain their functionality, e.g. linking with HSA and/or IL-17. In one embodiment, an antibody of the invention is a whole IgG that contains or has the heavy and light chains of SEQ ID NO: 131 (CA645 heavy chain with dsscFv CA497 (vHvL) inserted in framework 3) and SEQ ID NO: 1, respectively , or a variant or variants of any of them having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity, provided that they retain their functionality, e.g. binding to HSA and/or IL-17. In one embodiment, the antibody of the invention is a whole IgG that contains or has the heavy and light chains of SEQ ID NO: 132 (IgG4P heavy chain CA645 with dsscFv CA497 (vLvH) embedded in framework region 3) and SEQ ID NO: 1, respectively, or a variant or variants of any of them having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity, provided that they retain their functionality, for example, binding to HSA and/or IL-17.

In one embodiment, an antibody of the invention binds to both CD3 and CD28. In one embodiment, the antibody of the invention is a Fab fragment that binds both CD3 and CD28, in particular, the antibody containing the V domain binds to CD3, and the insert polypeptide is a dsscFv that binds CD28 i.e. . the framework antibody is a Fab fragment that binds CD3, and the second antibody inserted into the Fwk3 region of the V domain of the framework antibody is dsscFv that binds CD28. In one embodiment, the antibody of the invention is a Fab fragment that binds both CD3 and CD28, in particular the V-domain containing antibody binds to CD28, and the insert polypeptide is a dsscFv that binds CD3, i.e. e. the framework antibody is a Fab fragment that binds CD28, and the second antibody inserted into the Fwk3 region of the V domain of the framework antibody is dsscFv that binds CD3. In another embodiment, the antibody of the invention is a whole IgG that binds both CD3 and CD28, in particular the V-domain containing antibody binds to CD3 and the insert polypeptide is a dsscFv that binds CD28, i.e. . the framework antibody is a full IgG that binds CD3, and the second antibody inserted into the Fwk3 region of the V domain of the framework antibody is dsscFv that binds CD28. In another embodiment, the antibody of the invention is a whole IgG that binds both CD3 and CD28, in particular the V-domain containing antibody binds CD28, and the insert polypeptide is a dsscFv that binds CD3, i. the framework antibody is a full IgG that binds CD28, and the second antibody inserted into the Fwk3 region of the V domain of the framework antibody is dsscFv that binds CD3. In one embodiment, an antibody of the invention is a Fab fragment that contains or has the heavy and light chains of SEQ ID NO: 139 (anti-CD28 Fab heavy chain with anti-CD3 dsscFv embedded in framework 3) and SEQ ID NO: 136, respectively, or a variant or variants of any of them having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity, provided that they retain their functionality, for example, binding to CD3 and/or CD28. In another embodiment, an antibody of the invention is a whole IgG that contains or has the heavy and light chains of SEQ ID NO: 141 (anti-CD28 IgG4P heavy chain with anti-CD3 dsscFv embedded in framework 3) and SEQ ID NO: 136, respectively , or a variant or variants of any of them having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity, provided that they retain their functionality, e.g. binding to CD3 and/or CD28.

Illustrative sequences for use in the present invention include SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 , SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO : 76, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 , SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO : 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 , SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144. Exemplary combinations of heavy and light chains for use in Fab fragments of the present invention include SEQ ID NO: 54 and SEQ ID NO: 1, SEQ ID NO: 56 and SEQ ID NO: 1, SEQ ID NO: 58 and SEQ ID NO: 1, SEQ ID NO: 60 and SEQ ID NO: 1, SEQ ID NO: 61 and SEQ ID NO: 1, SEQ ID NO: 63 and SEQ ID NO: 1, SEQ ID NO: 64 and SEQ ID NO: 1, SEQ ID NO: 66 and SEQ ID NO: 1, SEQ ID NO : 68 and SEQ ID NO: 1, SEQ ID NO: 70 and SEQ ID NO: 1, SEQ ID NO: 73 and SEQ ID NO: 1, SEQ ID NO: 74 and SEQ ID NO: 1, SEQ ID NO: 75 and SEQ ID NO: 1, SEQ ID NO: 76 and SEQ ID NO: 1, SEQ ID NO: 93 and SEQ ID NO: 1, SEQ ID NO: 94 and SEQ ID NO: 1, SEQ ID NO: 95 and SEQ ID NO: 1, SEQ ID NO: 101 and SEQ ID NO: 1, SEQ ID NO: 129 and SEQ ID NO: 1, SEQ ID NO: 130 and SEQ ID NO: 1, SEQ ID NO: 139 and SEQ ID NO : 136, SEQ ID NO: 140 and SEQ ID NO: 133. Exemplary combinations of heavy and light chains for general IgG use of the present invention include SEQ ID NO: 131 and SEQ ID NO: 1, SEQ ID NO: 132 and SEQ ID NO: 1, SEQ ID NO: 141 and SEQ ID NO: 136, SEQ ID NO: 142 and SEQ ID NO: 133.

Linker sequence

In some embodiments, the insert polypeptide is linked directly to a portion of the framework region 3 of the antibody. By direct linkage, as used herein, is meant the fact that the N- and C-terminal amino acids of a heterologous insert polypeptide are linked via a peptide bond to the framework region 3 portion of the V domain of an antibody of the present invention.

In some embodiments, an antibody of the invention further comprises a linker sequence. In such embodiments, the linker sequence is preferably heterologous to both the antibody and the insert polypeptide, i.e. the linker sequence is not normally present in the native antibody sequence or insert polypeptide at that position.

In some embodiments, the linker sequence comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, or thirty-one amino acids connecting the N- and/or C-termini of the insert polypeptide to the framework region 3 region. In some embodiments, the linker sequence comprises one to thirty one, or five to thirty one, or six to twenty eight, or ten to twenty six, or fifteen to twenty six, or ten to twenty one, or five to ten amino acids linking the N- and/or C-terminus of the insert polypeptide to the framework region 3.

In some embodiments, the linker sequence contains no more than thirty amino acids connecting the N- and/or C-terminus of the insert polypeptide to the framework region 3 region.

In some embodiments, the linker at the N-terminus of the insert polypeptide is different in composition and/or length from the linker at the C-terminus of the insert polypeptide. In some embodiments, the linker at the N-terminus of the insert polypeptide is identical or similar in composition and/or length to the linker at the C-terminus of the insert polypeptide. In some embodiments, the linker sequence is derived from a human sequence. In some embodiments, the linker is a human linker. In some embodiments, the linker sequence is from a non-human sequence. In some embodiments, the implementation of the linker is from the sequence of the animal subfamily bovine. In some embodiments, the implementation of the linker is a linker from the sequence of the animal subfamily bovine.

Examples of suitable linker regions for linking the insert polypeptide to the framework region 3 portion of the V domain include, but are not limited to, flexible linker sequences and rigid linker sequences. Flexible linker sequences include those described in Huston et al., 1988, PNAS 85:5879-5883; Wright & Deonarain, Mol. Immunol., 2007, 44(11):2860-2869; Alfthan et al., Prot. Eng., 1995, 8(7):725-731; Luo et al., J. Biochem., 1995, 118(4):825-831; Tang et al., 1996, J. Biol. Chem. 271(26):15682-15686; and Turner et al., 1997, JIMM 205, 42-54 (see Table 1 below for representative examples).

Table 1 - Examples of flexible linker sequences

SEQID NO: SUBSEQUENCE eleven SGGGGSE 12 DKTHTS 13 (S)GGGGS 14 (S)GGGGSGGGGS 15 (S)GGGGSGGGGSGGGGS 16 (S)GGGGSGGGGSGGGGSGGGGS 17 (S)GGGGSGGGGSGGGGSGGGGSGGGGS 18 AAAGSG-GASAS 19 AAAGSG-XGGGS-GASAS 20 AAAGSG-XGGGSXGGGS-GASAS 21 AAAGSG-XGGGSXGGGSXGGGS-GASAS 22 AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 23 AAAGSG-XS-GASAS 24 PGGNRGTTTTRRPATTTGSSPGPTQSHY 25 ATTTGSSPGPT 26 ATTTGS - GS 27 EPSGPISTINSPPSKESHKSP 28 GTVAAPSVFIFPPSD 29 GGGGIAPSMVGGGGS thirty GGGGKVEGAGGGGGS 31 GGGGSMKSHDGGGGS 32 GGGGNLITIVGGGGS 33 GGGGVVPSLPGGGGS 34 GGEKSIPGGGGS 35 RPLSYRPPFPFGFPSVRP 36 YPRSIYIRRRHPSPSLTTT 37 TPSHLSHILPSFGLPTEN 38 RPVSPFTFPRLSNSWLPA 39 SPAAHFPRSIPRPPGPIRT 40 APGPSAPSHRSLPSRAFG 41 PRNSIHFLHPLLVAPLGA 42 MPSLSGVLQVRYLSPPDL 43 SPQYPSPLTLTLPPPHPSL 44 NPSLNPPSYLHRAPSRIS 45 LPWRTSLLPSLPLRRRP 46 PPLFAKGPVGLLSRSFFPP 47 VPPAPVVSLRSAHARPPY 48 LRPTPPRVRSYTCCPTP- 49 PNVAHVLPLLTVPWDNLR 50 CNPLLPLCARSPAVRTFP 77 KYQSGGSGMG 78 SGGSYTY 79 GGGSGGGG 80 GSSSSGS - GGG 81 SGGGG - S

(S) is optional in sequences 13-17. X stands for any remainder.

In specific embodiments, the antibody comprises linker sequences of SEQ ID NOS: 106 and 107.

Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQ ID NO:51), PPPP (SEQ ID NO:52), PPP, IPFTV (SEQ ID NO:82) and EYHGLQ (SEQ ID NO:104). The following examples include sequences derived from the CDRH3 extended stem of bovine antibodies (Wang et al., Cell, 2013, 153:1379-1393); preferred sequences include long linker sequences such as TSVHQETKKYQS (SEQ ID NO: 96) and SYTYNYEWHVDV (SEQ ID NO: 97) (containing both solvent-exposed and submerged residues from the bovine CDH3 stem); and short linker sequences such as ETKKYQS (SEQ ID NO: 98) and SYTYNYE (SEQ ID NO: 99) (containing only solvent exposed residues from the bovine CDH3 stem).

In some embodiments, variants of any of the linker sequences described herein can be used, for example, variants where one, two, three, four or more amino acid residues are substituted, inserted, or deleted relative to the original linker sequence. Linker sequence variants may also include repeat variants where the linker sequence is extended by repeating the linker sequence. In one embodiment, a preferred linker sequence to be repeated is SEQ ID NO: 13, which provides the motif: (G _m S) _n where m is 4 and n can be any integer (eg, 1, 2, or 3). In other embodiments, m may be a different integer from 1 to 10.

Whether the insert polypeptide is linked directly or via a linker sequence, in some embodiments, one or more amino acids in framework region 3 may be removed by linking the insert polypeptide. In some embodiments, the insert polypeptide is between amino acid residues 73 and 75 of the VH domain according to the Kabat numbering system and the amino acid at position 74 is deleted.

Pharmaceutical Compositions and Methods of Use

The antibodies of the invention and as described herein are useful in the treatment of diseases or disorders, including inflammatory diseases and disorders, immune diseases and disorders, fibrotic disorders, and cancers.

The terms "inflammatory disease" or "disorder" and "nominal disease or disorder" include rheumatoid arthritis, psoriatic arthritis, Still's disease, Muckle-Wells disease, psoriasis, Crohn's disease, ulcerative colitis, SLE (systemic lupus erythematosus), asthma, allergic rhinitis , atopic dermatitis, multiple sclerosis, vasculitis, type I diabetes mellitus, transplantation, and graft versus host disease.

The term "fibrotic disorder" includes idiopathic pulmonary fibrosis (IPF), systemic sclerosis (or scleroderma), renal fibrosis, diabetic nephropathy, IgA nephropathy, hypertension, end-stage chronic renal failure, peritoneal fibrosis (permanent ambulatory peritoneal dialysis), cirrhosis of the liver, associated with aging, macular degeneration (ARMD), retinopathy, cardiac reactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty, coronary artery bypass grafting, arthroplasty, and cataract surgery.

The term "cancer" includes a malignant new growth that originates from the epithelium, is found on the skin, or more commonly the lining of organs of the body, such as the breast, ovaries, prostate, lung, kidney, pancreas, stomach, bladder, or colon. Malignant tumors tend to infiltrate neighboring tissues and spread (metastasize) to other organs, such as the bone, liver, lung, or brain.

Thus, according to a further aspect of the invention, a pharmaceutical composition is provided which comprises an antibody of the invention together with one or more pharmaceutically acceptable carriers, excipients or diluents.

Also contemplated is the use of an antibody of the invention for the manufacture of a medicament for the treatment of a disease or disorder. Most preferably, the disease or disorder is an inflammatory disease or disorder.

Pharmaceutical compositions according to the invention may be in a form, for example, suitable for parenteral, subcutaneous or ophthalmic administration. When appropriate, for example if an antibody binds to albumin via its V domain, it may be desirable to preformulate an antibody of the invention with human serum albumin or recombinant serum albumin using any suitable method known in the art.

Where the pharmaceutical composition is a liquid, eg a solution or suspension, the composition may further comprise an albumin, eg human serum albumin, in particular a recombinant albumin such as recombinant human serum albumin. Suitable amounts may be in the range of less than 2% wt./mass. from the total composition, in particular, less than 1, 0.5 or 0.1% wt./mass. This may contribute to the stabilization of the antibody component in the formulation. The pharmaceutical composition may be lyophilized to be reconstituted later with an aqueous solvent. In one embodiment, a unit dose container, such as a vial containing a lyophilized antibody of the invention, is provided.

The antibodies of the invention can be formulated for parenteral administration by injection, for example by bolus injection or infusion. Compositions for injection may be present in unit dosage form, for example, in glass ampoules or in multi-dose containers, for example, in glass vials. Compositions for injection may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending agents, stabilizing agents, preservatives and/or dispersing agents. Alternatively, the active ingredient may be in the form of a powder for reconstitution with a suitable vehicle, eg, sterile pyrogen-free water, prior to use.

If desired, the compositions may be presented in a package or dispensing device which may contain one or more unit dosage forms containing the active ingredient. The package or dispenser may be accompanied by instructions for administration.

The antibody formats of the invention can be delivered dispersed in a solvent, for example in the form of a solution or suspension. They can be suspended in a suitable saline solution, such as saline or other pharmacologically acceptable solvent or buffer solution. Buffer solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0, 25 mg to 0.30 mg anhydrous citric acid and 0.45 mg to 0.55 mg sodium citrate in 1 ml of water so as to achieve a pH of approximately 4.0 to 5.0. You can use a suspension, for example, lyophilized antibody.

Therapeutic formulations of suspensions or solutions may also contain one or more excipients. Excipients are well known in the art and include buffers (eg, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (eg, serum albumin), EDTA, sodium chloride, liposomes. , mannitol, sorbitol and glycerin. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation is typically provided in a substantially sterile form using sterile manufacturing processes.

This may include producing and sterilizing by filtering the buffer solvent/solution used for formulation, aseptically suspending the antibody in a sterile buffer solvent solution, and dispensing the formulation into a sterile container by methods known to one of skill in the art.

The amount of an antibody of the invention required to prevent or treat a particular condition varies depending on the condition being treated. However, in general, daily dosages may be in the range of about 10 ng/kg to 1000 mg/kg, 100 ng/kg to 100 mg/kg, or 0.1 mg/kg to 10 mg/kg of body weight.

The present invention also relates to methods and compositions for delivering antibodies as described herein via gene therapy, in particular via an adeno-associated viral (AAV) vector.

Thus, the present invention relates to a pharmaceutical composition comprising a viral vector having a viral capsid and an artificial genome containing an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene containing a polynucleotide sequence encoding an antibody as described herein. description. ITR sequences can be used to package an artificial genome containing antibody-encoding polynucleotide sequences as described herein into a viral vector virion.

The transgene in the expression cassette is operably linked to expression control elements such as promoters that control the expression of the transgene in human cells.

The viral vector is preferably AAV-based viral vectors. Various AAV capsids are described in the art. Methods for producing vectors based on AAV are also widely described in the literature (for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US patent No. 7588772 B2). The source of AAV capsids can be selected from AAV that targets the desired tissue. For example, a suitable AAV may include, for example, AAV9 (US Patent No. 7906111; US 2011-0236353-A1), rh10 (WO 2003/042397) and/or hu37 (US 7906111B2; US20110236353). However, other AAVs may also be selected, including, for example, AAV1, AAV2, AAV-TT, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV.PHP.B (or variants thereof) and others.

Methods for obtaining and isolating AAV-based viral vectors suitable for delivery to an individual are known in the art (US7790449B2; US7282199B2; WO 2003/042397; WO 2005/033321, WO 2006/110689; and US7588772 B2).

A pharmaceutical composition containing a viral vector having a viral capsid and an artificial genome according to the invention is designed to allow expression of both heavy and light chains. The antibody heavy and light chain polynucleotide sequences of the present invention may be constructed as a single construct and may be separated, for example, by a cleavable linker or internal ribosome entry site (IRES) element, such that the heavy and light chains are expressed separately. In some embodiments, the cleavable linker is a self-cleaving furin/F2A linker (Fang et al., Nature Biotechnology 23: 584-590, 2005 and Fang et al. Mol Ther 15: 1153-9, 2007). For example, a furin/F2A linker can be included in an expression cassette to separate sequences encoding heavy and light chain polynucleotides.

In some other embodiments, the heavy and light chain polynucleotide sequences of the antibodies of the present invention are connected via a flexible, non-cleavable linker. In some embodiments, such a linker may be a flexible peptide linker, such as scFv-encoding linkers (consisting of Gly and Ser), such that the heavy and light chain domains move freely relative to each other.

The invention is further described with reference to the following examples, which are illustrative only and should in no way be construed as limiting the scope of the present invention.

EXAMPLES

General methodology

Paste in wireframe 3

In addition to the three CDR loops, the light and heavy chains of both conventional antibodies and camelid single chain VHHs have a fourth loop that is formed by framework region 3 (FIG. 1A). The Kabat numbering system defines framework 3 as heavy chain positions 66-94 and light chain positions 57-88.

Gene construction and transient cell expression

Genes encoding the V regions of the heavy and light chains, including variants with an insert in the frame region 3, were modeled and constructed using an automated synthesis approach (ATUM).

The genes were cloned into expression vectors containing DNA encoding the human Cκ region (Km3 allotype) and the γ1 heavy chain CH1 region, respectively. HEK293 cells were then cotransfected with heavy and light chain expression vectors and recombinant antibody/Fab fusion protein molecules were screened using an SPR binding assay.

The light chain V region or light chain of the CA645 Fab fusion proteins described in the examples below contained or had SEQ ID NO: 2 (CA645 VL domain (gL5)) or SEQ ID NO: 1 (CA645 light chain gL5), respectively . Alternative light chains or light chain V regions can be used, for example, light chain V regions containing the VL domain of SEQ ID NO: 108 or SEQ ID NO: 109.

Determination of Antibody/Fab Fusion Protein Binding Efficiency by Surface Plasmon Resonance

All surface plasmon resonance (SPR) experiments were performed at 25° C. on a Biacore 3000 system using running buffer at pH 7.4 containing 10 mM HEPES, 150 mM NaCl, 2 mM EDTA and 0.005% (v/v ) P20 (HBS-EP buffer).

A goat anti-human F(ab) ₂ polyclonal F(ab) ₂ fragment (Jackson Labs product code #109-006-097) was immobilized on the surface of CM5 sensor chips (GE Healthcare) by the amine attachment method. Briefly, the carboxymethyldextran surface was activated with a fresh 1:1 (v/v) mixture of 50 mM N-hydroxysuccinimide and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 5 minutes at a flow rate of 10 μl/min . Anti-human F(ab) ₂ in 10 mM acetate buffer, pH 5.0, was covalently bound to the chips at 50 μg/ml during treatment for 10 minutes at the same flow rate. Finally, the surface was inactivated by treatment for 10 minutes with 1 M ethanolamine. HCl, pH 8.5. Prepared the reference flow cell per chip by eliminating the protein from the above method.

Supernatants containing Fab-loop, Fab wild-type, or control supernatant insert constructs were diluted 1:5 in HPS-EP buffer prior to testing for their respective ligand or receptor binding.

Human serum albumin (HSA) was provided by Jackson ImmunoResearch (catalog number #009-000-051) and was diluted in HBS-EP buffer to 1000 nM. The following cytokines and receptors were obtained from R&D Systems Ltd.: IL-15, IL-15-Rα-hFc, IL-2, IL-2-sRβ, G-CSF, G-CSF-Rα, GM-CSF, GM-CSF -Rα, EPO, EPO-R, IL-6, LRP4(ecd)-hFc, LRP6(ecd)-hFc, lysozyme, KLK5. These proteins were usually diluted to 100 μg/ml and stored at -20°C. For experimental purposes, they were diluted just prior to use with HBS-EP buffer, typically to 50, 100, or 200 nM.

A typical SPR cycle included a flow rate of 10 μl/min with HBS-EP as running buffer and the diluted supernatant was injected for 1 min to capture the antibody/Fab fusion protein. This was followed by injection of 30 μl of diluted recognition ligand or receptor or buffer control. The subsequent dissociation phase was monitored for at least 3 minutes. In some experiments, an additional injection of 30 μl of the appropriate antibody-recognized antigen, HSA, was included in the case of fusion protein constructs using Fab CA645 as the polypeptide portion of the antibody. In some experiments, a specificity control loop was included where the recognized receptor/ligand (part of the insert in the antibody/Fab fusion protein) was premixed with a 2-fold molar excess of its binding partner. Each cycle ended with a regeneration step that included two injections of 10 μl of 40 mM HCl, between which an injection of 5 μl of 5 mM NaOH was performed.

Results were obtained 10 s before supernatant and ligand/receptor injections to determine capture of the fusion protein construct, and 5 s before and 15 s after the end of ligand/receptor injection to assess recognized ligand/receptor binding. The results obtained and the sensorgram data reflected the difference in number of resonance units (RU) between the polyclonal antibody capture flow cell and the reference flow cell.

The results and sensorgram data were processed by subtracting the appropriate control cycle from the ligand/receptor cycle to account for any slow dissociation of the fusion protein construct from the capture surface. In each experiment, the appropriate wild-type Fab supernatant was included to test for any non-specific ligand/receptor binding. If it was significant, it was subtracted from the values described above.

In the examples in which dissociation constants (K _D ) were evaluated, the method described above was modified by increasing the flow rate to 30 μl/min. After capturing the Fab construct, 90 μl of the recognized ligand or buffer control was injected followed by a 180 sec dissociation phase. Ligand binding was tested over a range of concentrations (0.1, 0.2, 0.5, 1, 2, 5 and 10 nM). The sensograms were corrected for drift by subtracting the ligand-0 sensogram and for non-specific interaction by subtracting the sensogram adjusted for wild-type Fab. Affinity constants were calculated from separate dissociation and association rate constants, which were adjusted to corrected sensorgrams using the Langmuir model of the BIAevaluation software (version 4.1.1). In the examples in which dissociation constants (K _D ) were evaluated, the method described above was modified by increasing the flow rate to 30 μl/min. After capturing the Fab construct, 90 μl of the recognized ligand or buffer control was injected followed by a 180 sec dissociation phase. Ligand binding was tested over a range of concentrations (0.1, 0.2, 0.5, 1, 2, 5 and 10 nM). The sensograms were corrected for drift by subtracting the ligand-0 sensogram and for non-specific interaction by subtracting the sensogram adjusted for wild-type Fab. Affinity constants were calculated from separate dissociation and association rate constants, which were adjusted to corrected sensorgrams using the Langmuir model of the BIAevaluation software (version 4.1.1).

EXAMPLE 1 Fab CA645 with IL-15 insert polypeptide

Interleukin 15 (IL-15) is a cytokine that plays a role in the immune response and functions to stimulate the proliferation of T cells and natural killer (NK) cells. It is structurally similar to IL-2 and its receptor shares chains with the IL-2 receptor.

The fusion protein was generated and expressed according to the methods described above for inserting the human IL-15 sequence into the framework region 3 of the CA645 Fab construct as shown below.

Human IL-15 sequence:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN (SEQ ID NO: 53)

Fab CA645 heavy chain with human IL-15 (bold and italics) embedded in framework 3, linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNKYQSGGSGMGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINSGGSYTYKNTV YLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 54)

In FIG. 2A shows Biacore analysis of binding efficiency for this CA645/IL-15 antibody fusion protein when tested with 100 nM IL-2-Rβ, IL-15-Rα (both of which are binding partners for IL-15) and 1000 nM HSA (lignad for CA645). As can be clearly seen from FIG. 2A, binding was observed for all three ligands, demonstrating that functionality is retained in both the CA645 Fab portion and the IL-15 graft in the framework region 3. FIG. 2B shows a simulated representation of the CA645/IL-15 _Fwk3 protein.

EXAMPLE 2 - CA645 Fab with IL-2 insert polypeptide

Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells that plays a key role in the normal immune response. IL-2 stimulates the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells.

The fusion protein was generated and expressed according to the methods described above for inserting the human IL-2 sequence into the framework region 3 portion of the CA645 Fab construct as shown below.

Human IL-2 sequence:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 55)

Fab CA645 heavy chain with human IL-2 (bold and italics) embedded in framework 3, linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGSGGGGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETAT IVEFLNRWITFCQSIISTLTGGGKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 56)

In FIG. 3A shows Biacore analysis of binding efficiency for this CA645/IL-2 antibody fusion protein when tested with 100 nM IL-2-Rβ (binding partner for IL-2), preceded by incubation with 200 nM IL-15 (competitive ligand), or without her. As shown in FIG. 3A, the observed binding to IL-2-Rβ is abolished in the presence of a competing ligand in molar excess, demonstrating that the observed binding to the IL-2 moiety in framework region 3 of the fusion protein is a true functional interaction, and is not non-specific. In FIG. 3B shows a simulated representation of the CA645/IL-2 _Fwk3 protein.

EXAMPLE 3 - Fab CA645 with G-CSF Insert Polypeptide

Granulocyte colony stimulating factor (G-CSF) is a cytokine produced by a number of different cell types (eg, endothelium, macrophages) that acts to stimulate the production of mature granulocytes from the bone marrow.

The fusion protein was generated and expressed according to the methods described above for inserting the human G-CSF sequence into the framework region 3 region of the CA645 Fab construct as shown below.

Human G-CSF sequence:

ATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO: 57)

Fab CA645 heavy chain with human G-CSF (bold and italics) embedded in framework 3, Gly-Ser linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFAT TIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQPSGGGGKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC (SEQ ID NO: 58)

In FIG. 4A shows Biacore analysis of the binding efficiency of this CA645/G-CSF antibody fusion protein when tested with 100 nM G-CSF-Rα (binding partner for G-CSF), 1000 nM HSA (ligand for CA645), and as G-CSF-Rα and HSA. In FIG. 4A shows, along with other examples, that both the G-CSF and Fab portions of the fusion protein are functionally active and able to bind their respective binding partners. In FIG. 4A further shows that in the presence of both binding partners, the signal observed in the SPR curve is the sum of the individual binding curves, demonstrating that binding of both functional moieties can occur simultaneously. In FIG. 4B shows a simulated representation of the CA645/G-CSF _Fwk3 protein.

EXAMPLE 4 - CA645 Fab with GM-CSF Insert Polypeptide

GM-CSF is a cytokine that stimulates cells to produce granulocytes (neutrophils, eosinophils and basophils) and monocytes.

The fusion protein was generated and expressed according to the methods described above for inserting human and mouse GM-CSF sequences into framework region 3 of the CA645 Fab construct as shown below.

Human GM-CSF (hGM-CSF) sequence:

APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO: 59)

Fab CA645 heavy chain with human GM-CSF (bold and italics) embedded in framework 3, Gly-Ser linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGSGGGGAPARSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPPTPETSCATQIITFESFKENLKDFLLVIPF DCWEPVQEGGGKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 60)

Fab CA645 heavy chain with human GM-CSF (bold and italic) at the C-terminus of CH1, Gly-Ser linker underlined (for comparison purposes):

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (SEQ ID NO: 61)

Mouse GM-CSF sequence (mGM-CSF):

APTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK (SEQ ID NO: 62)

Fab CA645 heavy chain with mouse GM-CSF (bold and italic) embedded in frame 3, Gly-Ser linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGSGGGGAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFE (SEQ ID NO: 63)

Fab CA645 heavy chain with mouse GM-CSF (bold and italic) at the C-terminus of CH1, Gly-Ser linkers underlined (for comparison purposes):

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ (SEQ ID NO: 64)

In FIG. 5A and 6 are Biacore analysis of binding efficiency for CA645/hGM-CSF and CA645/mGM-CSF antibody fusion proteins (respectively) when tested with their recognized GM-CSF receptor (at 100 nM concentration). The results demonstrate that binding to GM-CSF is specific and depends on the presence of the insert polypeptide. In FIG. also shown is a single curve for the constant domain (CH1) fusion construct of the GM-CSF peptide, demonstrating that the insert in framework 3 functions in a comparable manner. In FIG. 5B shows a simulated representation of the CA645/GM-CSF _Fwk3 protein.

EXAMPLE 5 - Fab CA645 with EPO Insert Polypeptide

Erythropoietin (EPO) is a cytokine released by the kidney in response to cellular hypoxia that stimulates the production of red blood cells in the bone marrow.

The fusion protein was generated and expressed according to the methods described above to insert the human EPO sequence into the framework region 3 of the CA645 Fab construct as shown below.

EPO sequence:

(SEQ ID NO: 65)

Heavy chain Fab CA645 with human EPO (bold and italics) embedded in framework 3, Gly-Ser linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGSGGGGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAIS ( SEQ ID NO: 66)

In FIG. 7A shows Biacore analysis of binding efficiency for this CA645/EPO antibody fusion protein when tested with 50 nM EPO-R (EPO binding partner), with or without prior incubation with 500 nM soluble EPO (competitive ligand). In FIG. 7A shows, among other examples, that the EPO portion of the fusion is functionally active and able to bind to the receptor it recognizes. Moreover, the decrease in signal observed for prior incubation with EPO demonstrates the specificity of the interaction, while the unmodified Fab CA645 acts as a further control to demonstrate that the observed binding occurs with the EPO graft. In FIG. 7B shows a simulated representation of the CA645/EPO _Fwk3 protein.

EXAMPLE 6 - Antibody/Fab fusion protein with Fab CA645 with VHH6 insert

VHH6 is an engineered single domain camelid heavy chain antibody that binds a pooled epitope because it is able to bind the IL-6:gp80 complex, but not the individual components individually.

The fusion protein was generated and expressed according to the methods described above to insert the VHH6 sequence into framework region 3 of the CA645 Fab construct as shown below.

Camelid VHH6 sequence:

DVQFVESGGGSVHAGGSLRLNCATSGYIYSTYCMGWFRQAPGKEREGVAHIYTNSGRTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAIYYCAARPSIRCASFSATEYKDWGQGTQVTVSS (SEQ ID NO: 67)

Fab CA645 heavy chain with VHH6 (bold and italic) embedded in frame region 3, Gly-Ser linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSDVQFVESGGGSVHAGGSLRLNCATSGYIYSTYCMGWFRQAPGKEREGVAHIYTNSGRTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAIYYCAARPSIRCA (SEQ ID NO: 6) 8)

In FIG. 8A shows Biacore analysis of binding efficiency for this CA645/VHH6 antibody fusion protein when tested with 100 nM IL-6 (negative control for VHH6 because VHH6 only binds complex), 100 nM IL-6/gp80 fusion construct (binding partner for VHH6), 1000 nM HSA (binding partner for CA645) and both IL-6 and HSA fusion together. In FIG. 8A shows, among other examples, that both parts of the antibody fusion protein, VHH6 and CA645, are functionally active as binding fragments and are able to bind their respective binding partners. In FIG. 8A further shows that in the presence of both binding partners, the signal observed in the SPR curve is the sum of the individual binding curves, demonstrating that binding to both functional moieties can occur simultaneously. In FIG. 8B shows a simulated representation of the CA645/VHH6 _Fwk3 protein.

EXAMPLE 7 - Antibody/Fab fusion protein with Fab CA645 with VHH15 insert

VHH15 is an engineered single domain camelid heavy chain antibody that recognizes and binds IL-6.

The fusion protein was generated and expressed according to the methods described above to insert the VHH15 sequence into framework region 3 of the CA645 Fab construct as shown below.

Camelid VHH15 sequence:

QVQLVESGGGSVQAGGSLRLSCVAASGYTGCTYDMRWYRQAPGKEREFVSGIDSDGRATYADSVKGRFTISQSNAKIAVYLQMDSLKLEDTAMYYCNLQCLRYPGEYYWGQGTQVTVSS (SEQ ID NO: 69)

Heavy chain Fab CA645 with VHH15, a camelid domain-based anti-IL-6 antibody (bold and italic) embedded in framework 3, Gly-Ser linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSQVQLVESGGGSVQAGGSLRLSCVAASGYTGCTYDMRWYRQAPGKEREFVSGIDSDGRATYADSVKGRFTISQSNAKIAVYLQMDSLKLEDTAMYYCNLQCLRYPGEYYWG QGTQVTVSSGGGGSGGGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 70)

In FIG. 9A shows the Biacore analysis of binding efficiency for this CA645/VHH15 antibody fusion protein when tested with 100 nM IL-6 (ligand for VHH15), 1000 nM HSA (ligand for CA645), and both IL-6 and HSA. In FIG. 9A shows, among other examples, that both portions of the antibody fusion protein, VHH15 and Fab, are functionally active as binding fragments and are capable of binding their respective binding partners. In FIG. 9A further shows that in the presence of both binding partners, the signal observed in the SPR curve is the sum of the individual binding curves, demonstrating that binding to both functional moieties can occur simultaneously. In FIG. 9B shows a simulated representation of the CA645/VHH15 _Fwk3 protein.

EXAMPLE 8 - Fab CA645 with Sclerostin Insert Polypeptide

Sclerostin is a product of the human SOST gene and is involved in bone formation, mainly being expressed in osteocytes. Sclerostin functions as a regulatory protein in the Wnt signaling cascade by binding to LRP-type co-receptors.

Fusion proteins were generated and expressed according to the methods described above to insert the central or entire sclerostin sequence into the framework region 3 region of the CA645 Fab construct as shown below.

Mature full length human sclerostin sequence:

QGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY (SEQ ID NO: 71)

Human sclerostin central domain sequence:

CRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC (SEQ ID NO: 72)

Fab CA645 heavy chain with human sclerostin central domain (bold and italics) embedded in framework 3, linkers from one S underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 73)

Fab CA645 heavy chain with human sclerostin central domain (bold and italics) embedded in framework 3, Gly-Ser linkers (2X G ₄ S) underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCSGGGGGSGGGSKNTVYLQMNSLRAEDTAV YYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 74)

Fab CA645 heavy chain with human sclerostin central domain (bold and italics) embedded in framework 3, rigid linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSIPFTVCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCEYHGLQKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPY FDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 75)

Fab CA645 heavy chain with mature full-length human sclerostin (bold and italics) embedded in framework 3, Gly-Ser linkers (1X G ₄ S) underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSQGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRAQRVQLLC PGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAYSGGGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSC (SEQ ID NO: 76)

In FIG. 10A and 11 are Biacore analysis of the efficacy of this CA645/sclerostin antibody fusion protein when tested as a 1:5 diluted supernatant for binding to the immobilized extracellular domains of LRP6 and LRP4, respectively (binding partners for sclerostin). This experiment also explored the effects of various linker region designs. It was noted that all of the constructs tested showed binding to LRP6 and LRP4. In FIG. 10B shows a simulated representation for the CA645/Sclerostin _core-Fwk3 protein.

EXAMPLE 9 - CA645 Fab with IL-10 Insert Polypeptide (IL-10 Dimer)

Interleukin 10 (IL-10), also known as human cytokine synthesis incubation factor (CSIF), is an anti-inflammatory cytokine. It mainly functions as a homodimer.

The fusion protein was generated and expressed according to the methods described above for inserting two human IL-10 sequences into framework region 3 of the CA645 Fab construct as shown below.

Human IL-10 monomer sequence:

SEQ ID NO: 100 )

Fab CA645 heavy chain with two human IL-10 monomers (bold and italics) embedded in framework 3, linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGSNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKEPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFC PGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAGEYKGGGGSGGGGSGGGGSNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKEPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFC PGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGGGGSGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC (SEQ ID NO: 101)

In FIG. 12A shows Biacore analysis of binding efficiency for this CA645/IL-10 antibody fusion protein when tested with 100 nM IL-10 receptor (binding partner for IL-10), preceded by incubation with 200 nM IL-10 (competing ligand). or without it. As shown in FIG. 12A, the binding observed for the IL-10 receptor was abolished in the presence of a competing ligand in molar excess, demonstrating that the binding observed with the IL-10 portion of framework region 3 of the fusion protein is a true functional interaction and is not non-specific. This result also indicates that the inserted IL-10 monomers are also capable of forming a functional dimer in the framework construct 3. FIG. 12B shows a simulated representation of the CA645/IL-10 _Fwk3 protein.

EXAMPLE 10 - Antibody Framework 3 Insert Designs with Other Fabs

Fusion proteins were generated and expressed according to the methods described above for inserting a human IL-15 sequence into the framework region 3 of the CA497 and CA240 Fab constructs as shown below.

Anti-IL-17A Fab CA497 heavy chain sequence (Fab, CH1 from IgG1; variable domain underlined; CDR in bold and italics)

EVQLVESGGGLVKPGGSLRLSCAASGVIFSDYMAWVRQAPGKGLEWVASINFNADISYYRESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 125)

Heavy chain Fab CA497 against IL-17A with human IL-15 (bold and italics) embedded in framework 3, Gly-Ser linkers underlined:

EVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYMAWVRQAPGKGLEWVASINFNADISYYRESVKGRFTISRDDSGGGSGGGGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINGGGKNT LYLQMNSLKTEDTAVYYCTTDANRQNYDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 83)

Light chain sequence of Fab CA497 against IL-17A:

AIQLTQSPSSLSASVGDRVTITCKASESVSSSMYSYMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 84)

Anti-IL-6 Fab CA240 heavy chain sequence (Fab, CH1 from IgG1; variable domain underlined; CDRs in bold and italics):

EVQLVESGGGLVQPGGSLRLSCAASGFNFNDYFMNWVRQAPGKGLEWVAQMRNKNYQYGTYYAESLEGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARESYYGFTSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 126)

Anti-IL-6 Fab CA240 heavy chain with human IL-15 (bold and italics) embedded in framework 3, Gly-Ser linkers underlined:

EVQLVESGGGLVQPGGSLRLSCAASGFNFNDYFMNWVRQAPGKGLEWVAQMRNKNYQYGTYYAESLEGRFTISRDDSGGGSGGGGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINGGGK NSLYLQMNSLKTEDTAVYYCARESYYGFTSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:85)

Light chain sequence of Fab CA240 against IL-6:

DIQMTQSPSSLSASVGDRVTITCQASQDIGISLSWYQQKPGKAPKLLIYNANNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNSAPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 86)

In FIG. 13 and 14 show Biacore analysis of binding efficiency for antibody fusion proteins CA497/IL-15 and CA240/IL-15 (respectively) when tested with 100 nM IL-2-Rβ (binding partner for IL-15), with prior incubation with or without IL-15 (competing ligand). As shown in both experiments, the observed binding to IL-2-Rβ was abolished in the presence of a competing ligand in molar excess, demonstrating that the observed binding to the IL-15 portion of the framework region 3 of the fusion proteins is a true functional interaction and is not non-specific.

EXAMPLE 11 - Fab constructs with VHH embedded in frame region 3

HyHEL5 is an anti-lysozyme Fab with the following sequences:

HyHEL5 heavy chain:

EVQLQQSGAELMKPGASVKISCKASGYTFSDYWIEWVKQRPGHGLEWIGEILPGSGSTNYHERFKGKATFTADTSSSTAYMQLNSLTSEDSGVYYCLHGNYDFDGWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 87)

Light chain HyHEL5:

DIVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGTSYSLTISSMETEDAATYYCQQWGRNPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC (SEQ ID NO: 88)

D3L11 is an anti-lysozyme VHH with the following sequence:

D3L11:

DVQLVESGGGSVQAGGSLRLSCAASGSTDSIEYMTWFRQAPGKAREGVAALYTHTGNTYYTDSVKGRFTISQDKAKNMAYLRMDSVKSEDTAIYTCGATRKYVPVRFALDQSSYDYWGQGTQVTVSS (SEQ ID NO: 89)

The following occult constructs were generated to test whether insertion of a second antibody molecule into framework region 3 of the first antibody, where antibodies binding to the same target antigen would have a higher affinity for the antigen than each of the individual antibodies.

Anti-lysozyme HyHEL5 light chain with VHH D3L11 anti-lysozyme (bold) embedded in framework 3, Gly-Ser linkers underlined:

DIVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGGGGGSDVQLVESGGGSVQAGGSLRLSCAASGSTDSIEYMTWFRQAPGKAREGVAALYTHTGNTYYTDSVKGRFTISQDKAKNMAYLRMDSVKSEDTAIYTCGATRKYVPVRFALDQSSYDYWGQGTQ VTVSSGGGGSGGGGSGGGGSTSYSLTISSMETEDAATYYCQQWGRNPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90)

Anti-lysozyme HyHEL5 light chain with VHH15 anti-IL-6 (bold) embedded in framework 3, Gly-Ser linkers underlined:

DIVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGSGGGGSQVQLVESGGGSVQAGGSLRLSCVAASGYTGCTYDMRWYRQAPGKEREFVSGIDSDGRATYADSVKGRFTISQSNAKIAVYLQMDSLKLEDTAMYYCNLQCLRYPGEYYWGQGTQVTVSSGGG GSGGGGSGGGGSTSYSLTISSMETEDAATYYCQQWGRNPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 91)

CA645 light chain with anti-lysozyme VHH D3L11 (bold) embedded in framework 3, Gly-Ser linkers underlined:

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGSTDSIEYMTWFRQAPGKAREGVAALYTHTGNTYYTDSVKGRFTISQDKAKNMAYLRMDSVKSEDTAIYTCGATRKYVPVRFALDQSSYDYWG QGTQVTVSSGGGGSGGGGSGGGGSTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92)

The SPR curves for the dilution series of each of the constructs when tested for lysozyme binding are shown in FIG. 15-17. From the subsequent analysis using the methods described above, the following table shows the binding affinity for lysozyme calculated for each construct:

Table 2 - Binding affinity for antibody constructs

Design Fab specificity Specificity of VHH Binding affinity for lysozyme (K _D ) Antibody: Fab HyHEL5
Wireframe Insert 3: VHH D3L11 lysozyme lysozyme 2.6× ^10-11M Antibody: Fab HyHEL5
Wireframe Insert 3: VHH15 lysozyme IL-6 3.1×10 ^-10M Antibody: Fab CA645
Wireframe Insert 3: VHH D3L11 Human serum albumin lysozyme 4.8× ^10-10M

As clearly seen from the above results, the binding affinity for lysozyme increases (ie, a decrease in K _D is observed) for the construct where both the Fab and the VHH insert bind to lysozyme. In FIG. 18 illustrates how this can happen at the molecular level. This also suggests that the length of the linker may be important to allow for multivalent interactions on the same target molecule in such antibodies.

EXAMPLE 12 - CA645 Fab with IL-15 Insert Polypeptide Using Linkers from Animals of the Bovine Subfamily

Received variants of the protein CA645/IL-15 _Fwk3 described in example 1, where the linkers were replaced with other linkers, including linkers from animals of the bovine family, and tested their effects on binding. In addition to the original heavy chain construct (SEQ ID NO: 54), additional constructs were generated as shown below:

Heavy chain Fab CA645 with human IL-15 (bold and italics) embedded in framework 3 with long bovine linker (underlined)

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSTSVHQETKKYQSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINSYTYNYE (SEQ ID NO: 93)

Heavy chain Fab CA645 with human IL-15 (bold and italic) embedded in framework region 3 with short bovine linker (underlined)

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSETKKYQSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINSYTYNYEKNTVYL QMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 94)

Heavy chain Fab CA645 with human IL-15 (bold and italics) embedded in framework 3 with 2xG ₄ S linker (underlined)

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINGGGGSG GGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 95)

In FIG. 19 shows the Biacore binding assay for the CA645/IL-15 _Fwk3 constructs described above when tested with 100 nM IL-2-Rβ, and compared against the construct of Example 1 (parent linker) and wild type CA645 Fab that lacks the IL- 15. It should be noted that all of the constructs tested demonstrated successful binding.

EXAMPLE 13 Fab CA645 fragment with a dsscFv embedded in framework 3

Engineering of genes

Two antibodies of the invention were generated and expressed according to the methods described above to insert anti-IL17A dsscFv CA497 (vHvL or vLvH) into framework region 3 of the CA645 Fab fragment (framework antibody) as shown below. The resulting antibodies can be named CA645 Fab-Fwk3 CA497 dsscFv (vHvL) or CA645 Fab-Fwk3 CA497 dsscFv (vLvH), respectively. As controls received dsscFv CA497 and Fab CA645.

The genes encoding the V regions of the light and heavy chains, including variants with an insertion into the framework region 3, were modeled and constructed using an automated synthesis (ATUM) approach. Light chain V region genes (eg, encoding the VL domain of CA645 SEQ ID NO: 2) were cloned into expression vectors containing DNA encoding the human Cκ region (Km3 allotype). The heavy chain V region genes were cloned into expression vectors containing DNA encoding the IgG1 CH1 region.

dsscFv CA497 (vHvL)

EVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYMAWVRQAPGKCLEWVASINFNADISYYRESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCKASESVSSSSYMHWYQQKPGKAPKLLI YRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGCGTKVEIK (SEQ ID NO: 127)

dsscFv CA497 (vLvH)

AIQLTQSPSSLSASVGDRVTITCKASESVSSSMYSYMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYMAWVRQAPGKCLEWVASINFNADISYYRESVKGR FTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWFAYWGQGTLVTVSS (SEQ ID NO: 128)

Fab CA645 heavy chain with dsscFv CA497 (vHvL) (bold and italic) embedded in framework 3, linkers underlined

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYMAWVRQAPGKCLEWVASINFNADISYYRESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWF AYWGQGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCKASESVSSSMYSYMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGCGTKVEIKGGGGSGGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 129)

Fab CA645 heavy chain with dsscFv CA497 (vLvH) (bold and italics) embedded in frame 3, linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCKASESVSSSMYSYMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGCGTKVEIKG GGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYYMAWVRQAPGKCLEWVASINFNADISYYRESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWFAYWGQGTLVTVSSGGGGSGGGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 130)

Transient expression in cells

The engineered Fab fragments were transiently expressed either in HEK-293 cells using the ExpiFectamine 293 transfection kit (Life Technologies, according to the manufacturer's instructions) or in CHO-S XE cells derived from the CHO-K1 cell line (Cain K et al, A CHO cell line engineered to express XBP1 and ERO1-Lα has increased levels of transient protein expression Biotechnology Progress 2013;29:697-706), using ExpiFectamine CHO transfection kit (Life Technologies, according to manufacturer's instructions). HEK-293 cells were used for small scale expression (2 ml) to generate antibody constructs for direct analysis using SPR. CHO-S XE cells were used for large scale expression (200 ml) to generate antibody constructs for purification prior to further characterization.

cleaning

Affinity chromatography was used to purify Fab fragments from culture supernatants. Supernatants containing antibodies were passed through a HiTrap protein G column (GE Healthcare). After a washing step with phosphate buffered saline (PBS), pH 7.4, the bound material was eluted using a gradient of PBS, pH 7.4 against increasing percentage by volume of 0.1 M phosphate citrate buffer, pH 2.6. The eluate was neutralized with 2 M Tris-HCl (pH 8). Fractions were pooled, quantified by absorbance at 280 nm, and concentrated using Amicon Ultra centrifuge filters (Merck Millipore). To isolate the monomer fractions of the constructs, size exclusion chromatography was used on a HiLoad 16/60, Superdex 200 (GE Healthcare) column equilibrated with PBS, pH 7.4. Fractions containing the monomeric Fab were pooled, quantified, concentrated and stored at 4°C.

UPLC (Ultra High Performance Liquid Chromatography):

2 μg of sample was injected onto a SEC BEH200 column pre-equilibrated with PBS, pH 7.4 and run for 10 minutes. Fluorescence intensity was recorded against elution time and peak areas were manually selected in the EMpower software.

UPLC elution profiles were obtained and analyzed. The peaks looked >98% monomeric (99.42% for CA645 Fab-Fwk3 CA497 dsscFv (vLvH), 99.51% for CA645 Fab-Fwk3 CA497 dsscFv (vHvL)). Controls dsscFv CA497 and Fab CA645 showed 99.99% and 98.43% multimeric forms, respectively (data not shown). Thus, the purified proteins were homogeneous and lacked multimeric forms or aggregates.

Molecular stability

Molecular stability was determined by analyzing the melting point (Tm) (unfolding index) by differential scanning calorimetry (DSC). Thermograms were obtained on an automated MicroCal VP DSC (Malvern Panalytical) at 0-100°C, using 1°C/min steps, with a pre-incubation scan time of 15 min, a filtration period of 5 s, and passive feedback. Buffer data was subtracted from the data before manually subtracting baseline and correcting for concentration. The thermograms were fitted to a non-2-state model in Origin7.0.

The results are shown in FIG. 20 (DSC curves; vertical axis: (Cp) specific heat; horizontal axis: temperature in degrees Celsius C (°C)) and in Table 3 below. dsscFv CA497 and Fab CA645 showed single transitions at 58.40°C and 83.57°C, respectively. When combined in Fab-Fwk3 format with dsscFv insert, the Tm of dsscFv CA497 is nearly identical (2-3°C difference) and Fab CA645 has a Tm variation of approximately 4-5°C. Thus, pairing of dsscFv CA497 and Fab CA645 in this antibody format does not significantly affect the conformational stability of each of the domains.

Table 3 - Transition temperatures for unwrapping

Sample Tm1 (°C) Tm2 (°C) Fab CA645 83.57 dsscFv CA497 58.40 Fab CA645 heavy chain with dsscFv CA497 (vLvH) built into Fwk3 55.59 78.64 Fab CA645 heavy chain with dsscFv CA497 (vHvL) built into Fwk3 55.41 78.73

Neutralization analysis

The efficacy of IgG4 antibody CA497, CA645 Fab-Fwk3 CA497 dsscFv (vHvL) and CA645 Fab-Fwk3 CA497 dsscFv (vLvH) against recombinant human IL-17A in Hela cells was tested in the presence or absence of HSA. Hela cells were obtained from the ATCC cell bank (ATCC CCL-2). Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, gentamicin, and glutamine. 1×10 ⁴ cells were seeded in 96-well flat bottom tissue culture plates. Cells were incubated overnight and washed once in assay buffer. Human IL-17A (25 ng ml ^-1 ) was incubated in the presence of a fixed concentration of human TNF-α, and this mixture was pre-incubated with the test antibody. Cytokines and antibody were then added to Hela cells to stimulate the cells, which were incubated overnight. In the Hela cell line, IL-17A acts synergistically with TNF-alpha to induce the production of IL-6, which can be quantified, for example, by ELISA. The production of IL-6 in the cell culture supernatant is proportional to the amount of IL-17A added to the cells. Human IL-6 levels were measured by ELISA and quantified by comparison with known standard human IL-6 concentrations. In this assay, inhibition of IL-6 secretion correlates with inhibition of IL-17A.

The inhibition curves are shown in Fig. 21 (horizontal axis: antibody (inhibitor) concentration in ng/mL, vertical axis: inhibition of IL-6 secretion (%)) and indicates that the parent IgG4 antibody CA497 and both antibody fusion constructs effectively neutralized recombinant human IL-17A. In addition, the results indicate that the anti-IL-17 dsscFv retained functionality when inserted into the anti-HSA Fwk3 Fab framework. The EC50 values calculated from these experiments (Table 4 below) indicated that the CA645 Fab-Fwk3 CA497 dsscFv (vLvH) and CA645 Fab-Fwk3 CA497 dsscFv (vHvL) antibody provided an EC _{50 of} 10.5 ng/mL and 10.6 ng /ml, respectively, against recombinant human IL-17A (25 ng.ml ^-1 ) and that the presence of HSA did not affect the effectiveness of neutralization (EC ₅₀ 10.8 and 8.7 ng/ml, respectively, in the presence of HSA). Also, the CA645 Fab-Fwk3 CA497 dsscFv constructs had a neutralization efficiency comparable to one of the parent IgG4 antibodies, CA497.

Table 4. EC ₅₀ values for neutralization of IL-17A in the presence or absence of HSA

HSA (2.5%) IL-17A EC ₅₀ (ng/ml) IgG4 CA497 - 8.1 IgG4 CA497 + 7.6 CA645 Fab-Fwk3 CA497 dsscFv (vLvH) - 10.5 CA645 Fab-Fwk3 CA497 dsscFv (vLvH) + 10.8 CA645 Fab-Fwk3 CA497 dsscFv (vHvL) - 10.6 CA645 Fab-Fwk3 CA497 dsscFv (vHvL) + 8.7

EXAMPLE 14 Complete IgG CA645 with dsscFv inserted into framework 3

The fusion protein was generated and expressed as described above for inserting dsscFv (vH-vL) CA497 or dsscFv (vL-vH) CA497 into framework region 3 of the CA645 IgG4P construct as shown below. The resulting antibody fusion proteins can be referred to as CA645 IgG4P Fwk3-CA497 dsscFv (vHvL) and CA645 IgG4P Fwk3-CA497 dsscFv (vLvH).

Gene construction

genes coding for the heavy and light chain V regions, including framework 3 insertion variants, were modeled and constructed by an automated synthesis (ATUM) approach.

Light chain V region genes (eg, encoding the CA645 VL domain of SEQ ID NO: 2) were cloned into expression vectors containing DNA encoding the human Cκ region (Km3 allotype). The heavy chain V region genes were cloned into expression vectors containing DNA encoding the IgG4P constant regions for the full length antibody.

Heavy chain IgG4P CA645 with dsscFv (vHvL) CA497 (bold and italics) embedded in framework 3, linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYMAWVRQAPGKCLEWVASINFNADISYYRESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWF AYWGQGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCKASESVSSSMYSYMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGCGTKVEIKGGGGSGGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 131)

Heavy chain IgG4P CA645 with dsscFv (vLvH) CA497 (bold and italic) embedded in framework 3, linkers underlined:

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCKASESVSSSMYSYMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSWTAPRTFGCGTKVEIKG GGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGVIFSDYYYMAWVRQAPGKCLEWVASINFNADISYYRESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDANRQNYDWFAYWGQGTLVTVSSGGGGSGGGGSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 132)

Transient expression in cells

Antibodies were transiently expressed either in HEK-293 cells using the ExpiFectamine 293 transfection kit (Life Technologies, according to the manufacturer's instructions) or in CHO-S XE cells derived from the CHO-K1 cell line (Cain K et al, A CHO cell line engineered to express XBP1 and ERO1-Lα has increased levels of transient protein expression Biotechnology Progress 2013;29:697-706), using ExpiFectamine CHO transfection kit (Life Technologies, according to manufacturer's instructions). HEK-293 cells were used for small scale expression (2 ml) to generate antibody constructs for direct analysis using SPR. CHO-S XE cells were used for large scale expression (200 ml) to generate antibody constructs for purification prior to further characterization.

cleaning

Affinity chromatography was used to purify IgG from culture supernatants. Supernatants containing IgG were passed through a MabSelect SuRe column (GE Healthcare). After a washing step with phosphate buffered saline (PBS), pH 7.4, the bound material was eluted using a gradient of PBS, pH 7.4 against increasing percentage by volume of 0.1 M phosphate citrate buffer, pH 2.6. The eluate was neutralized with 2 M Tris-HCl (pH 8). Fractions were pooled, quantified by absorbance at 280 nm, and concentrated using Amicon Ultra centrifuge filters (Merck Millipore). SEC on a HiLoad 16/60, Superdex 200 (GE Healthcare) column equilibrated with PBS, pH 7.4 was used to isolate the monomeric fractions of the IgG-based constructs. Fractions containing the monomeric Fab were pooled, quantified, concentrated and stored at 4°C.

Simultaneous binding to IL-17 and HSA

To test the simultaneous binding of IL-17A and HSA to CA645 IgG4P Fwk3-CA497 dsscFv (vHvL), the latter was immobilized on a Biacore chip instead of goat polyclonal F(ab) ₂ using the same method described above. Three concentrations of both IL-17A and HSA were chosen to provide low, medium and high levels of binding to the immobilized antibody, in particular, they corresponded to stoichiometric ratios of ligand and antibody in the ranges from 0.15 to 0.3, from 0.4 to 0.6 and 0.65 to 0.75, respectively. In the case of IL-17A, the concentrations were 4, 20, and 100 nM, and in the case of HSA, the concentrations were 40, 200, and 1000 nM, respectively. The above low mid and high concentrations were tested by SPR as previously described in individual cycles either as single lignads or as IL17A/HCC mixtures and corrected by cycle subtraction for buffer control. The results are shown in FIG. 22 and show that CA645 IgG4P Fwk3-CA497 dsscFv (vHvL) binds both IL-17A and HSA in a concentration dependent manner. In FIG. 22 further shows that in the presence of both binding partners, the signal is additive, demonstrating that binding to both IL-17A and HSA can occur simultaneously.

EXAMPLE 15 - Antibodies of the invention that bind to both CD3 and CD28

Gene construction

Antibodies were generated and expressed according to the methods described above to insert anti-CD3 dsscFv (vH-vL) into framework region 3 of an anti-CD3 or anti-CD28 framework antibody (IgG4P or Fab fragment) as shown in Table 5 below. antibody constructs according to the invention may be referred to as anti-CD28 Fab Fwk3-anti-CD3 dsscFv (LC SEQ ID NO: 136, HC SEQ ID NO: 139), anti-CD3 Fab Fwk3-anti-CD3 dsscFv (LC SEQ ID NO: 133 , HC SEQ ID NO: 140), anti-CD28 IgG4P Fwk3-anti-CD3 dsscFv (LC SEQ ID NO: 136, HC SEQ ID NO: 141), anti-CD3 IgG4P Fwk3-anti-CD3 dsscFv (LC SEQ ID NO: 141) : 133, HC SEQ ID NO: 142).

Table 5 - Antibody constructs

Framework antibody Antigenic specificity (framework V-domain) Antigenic specificity (dsscFv built into Fwk3) Subsequence fab CD3 No Light chain SEQ ID NO: 133
Heavy chain SEQ ID NO: 134 fab CD28 No Light chain SEQ ID NO: 136
Heavy chain SEQ ID NO: 137 fab CD28 CD3 Light chain SEQ ID NO: 136
Heavy chain SEQ ID NO: 139 fab CD3 CD3 Light chain SEQ ID NO: 133 Heavy chain SEQ ID NO: 140 IgG4P CD3 No Light chain SEQ ID NO: 133
Heavy chain SEQ ID NO: 135 IgG4P CD28 No Light chain SEQ ID NO: 136
Heavy chain SEQ ID NO: 138 IgG4P CD28 CD3 Light chain SEQ ID NO: 136
Heavy chain SEQ ID NO: 141 IgG4P CD3 CD3 Light chain SEQ ID NO: 133
Heavy chain SEQ ID NO: 142

Anti-CD3 antibody light chain, muromonab (humanized OKT3) (variable domain underlined; CDR in bold and italics):

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133)

Fab heavy chain against CD3, CH1 from IgG1, muromonab (humanized OKT3) (variable domain underlined; CDR in bold and italics):

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 134)

Anti-CD3 IgG4P heavy chain, muromonab (humanized OKT3) (variable domain underlined; CDR in bold and italics):

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 135)

Anti-CD28 antibody light chain, teralizumab (variable domain underlined; CDR in bold and italics):

DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136)

Fab heavy chain against CD28, CH1 from IgG1, teralizumab (variable domain underlined; CDR in bold and italics):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 137)

Anti-CD28 IgG4P heavy chain, teralizumab (variable domain underlined; CDR in bold and italics):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 138)

Anti-CD28 Fab heavy chain with anti-CD3 dsscFv (vHvL) (bold and italics) embedded in frame 3, linkers underlined:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVY FCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIKGGGGSGGGGSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFD VWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 139)

Anti-CD3 Fab heavy chain with dsscFv (vHvL) against CD3 (bold and italic) embedded in frame 3, linkers underlined:

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVY FCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIKGGGGSGGGGSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLD YWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 140)

Anti-CD28 IgG4P heavy chain with dsscFv (vHvL) against CD3 (bold and italic) embedded in framework 3, linkers underlined:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVY FCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIKGGGGSGGGGSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFD VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 141)

Anti-CD3 IgG4P heavy chain with dsscFv (vHvL) against CD3 (bold and italic) embedded in framework 3, linkers underlined:

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVY FCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIKGGGGSGGGGSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLD YWGQGTPVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 142)

UPLC (Ultra High Performance Liquid Chromatography):

2 µg of sample was injected onto a SEC BEH200 column pre-equilibrated with PBS, pH 7.4 and run for 10 min. Fluorescence intensity was recorded against elution time and peak areas were selected manually in the EMpower software.

UPLC elution profiles were obtained and analyzed. Peaks appeared >98% monomeric (anti-CD3 IgG4P Fwk3-anti-CD3 dsscFv: 99.02%, anti-CD3 Fab Fwk3-anti-CD3 dsscFv: 99.79%, anti-CD28 Fab Fwk3-anti-CD3 dsscFv : 99.48%) except for a lower molecular weight arm of 12.12% in anti-CD28 IgG4P Fwk3-anti-CD3 dsscFv. There was no evidence of multimerization in any of the formats.

Molecular stability

Molecular stability was determined by melting point (Tm) (unfolding index) analysis by differential scanning calorimetry (DSC) as described above. The DSC curves are shown in Fig. 23 (A) and (B) and the Tm values are shown in Tables 6 and 7 below.

Table 6 - Fab-Fwk3-dsscFv

Antibody of the invention Tm1 (°C) Tm2 (°C) anti-CD28 Fab Fwk3-anti-CD3 dsscFv 70.16 74.73 anti-CD3 Fab Fwk3-anti-CD3 dsscFv 70.04

DSC curves for anti-CD3 Fab Fwk3-anti-CD3 dsscFv showed a single unfolding transition at 70.04°C. In contrast, the unfolding of a construct containing an anti-CD28 construct cannot be accurately described by a single transition, but better fits a non-2-state model with two transitions; the lowest of which can be attributed to the dsscFv component. Remarkably, the results demonstrate that incorporation of Fab at the position of framework region 3 resulted in stable constructs, since the values of 65°C-75°C are comparable to those for typical IgG.

Table 7- IgG4P-Fwk3-dsscFv

Antibody of the invention Tm1 (°C) Tm2 (°C) Anti-CD28 IgG4P Fwk3-anti-CD3 dsscFv 68.97 73.28 Anti-CD3 IgG4P Fwk3-anti-CD3 dsscFv 68.55

DSC curves for anti-CD3 IgG4P Fwk3-anti-CD3 dsscFv showed a single unfolding transition at 68.55°C. In contrast, the unfolding of constructs containing a construct against CD28 cannot be accurately described by a single transition, but better fits a non-2-state model with two transitions; the lowest of which can be attributed to the dsscFv component. Remarkably, the results demonstrate that incorporating IgG into the framework region 3 position resulted in stable constructs, as values of 65°C-75°C are typical for IgG-like molecules.

Evaluation of Fab and IgG4P Constructs with Framework 3 Inserts in Functional Analysis with Human PBMC

The constructs were tested for their ability to stimulate T cell activation in peripheral blood mononuclear cell (PBMC) culture according to the method described below.

Reagents

Antibodies used to detect CD4 and CD8 T cells, discriminate between live and dead cells and determine the CD69 activation marker, and were obtained from BD Biosciences with fluoroforms FITC, PE, BV421, and Near Infrared, respectively.All cell culture was performed in RPMI1640 (Thermo) plus 5% heat-inactivated human AB serum (Sigma) and 2 mM glutamine (Thermo).All staining by flow cytometry was performed using standard flow cytometry buffer containing Dulbecco's PBS (Thermo) plus 1% bovine serum albumin (Sigma), 2 mM EDTA (Sigma) and 0.1% sodium azide (Sigma), human OKT3 (Ebioscience) and T cell Transact (Miltenyi ), a colloidal suspension of anti-CD3 and CD28 antibodies All flow cytometric data were obtained using iQUEplus (Sartorius) T cells were identified using Forecyt software (Sartorius) and data were imported into GraphPad Prism 7 (GraphPad) for data conversion and graphical presentation .

Way

The antibody constructs in Table 5 were diluted starting at 25 μg/ml in eight 1 to 5 dilution steps. In a subsequent analysis step, all of these values were converted to nM concentrations to ensure equal comparisons of all constructs. In 96-well "U" bottom tissue culture plates, 1.5 x 10 ⁵ PBMC (from two different donors, anonymously referred to as donors 1 and 2) were mixed with diluted antibodies. OKT3 (5 µg/ml), Transact (CD3/CD28 diluted 1:100) and medium alone were the reference controls for the experiment. After mixing, the plates were incubated at 37 degrees Celsius for 24 hours. After that, the plates were cooled on ice and centrifuged (all subsequent centrifugation was carried out at 500g for 5 min at 4 degrees Celsius) and the supernatant was removed from each well. The plates were washed again, the supernatant was aspirated and 20 µl of the antibody cocktail was added to each well (all antibodies were diluted 1:20 in flow cytometry buffer and 1 µl of live/dead dye was added per 1 ml of the cocktail). The plates were left on ice in the dark for 1 hour. Thereafter, the plates were washed an additional two times and the supernatant was removed from all wells. At the end, the plates were resuspended in 20 μl of flow cytometry buffer and at least 10,000 cells per well were obtained.

results

The results are shown in FIG. 24 (A-D; the vertical axis represents the expression level of CD69 (MFI) and the horizontal axis represents the concentration of the antibody construct in nM). The experiment was considered successful because the positive control (OKT3) induced the early activation marker CD69 on both CD4 and CD8 T cells. The minimum (cells only) level of activation was determined by the cell-only control. As can be seen from FIG. 24(A) and (B), CD69 induction on CD4 T cells for the constructs tested was comparable between donors.

Antibodies of the invention containing CD3 and CD28 (either in Fab or IgG4P format) were the most potent activators of CD4 T cells. As expected in this assay, Fc containing constructs can also confer additional activity via Fc receptors on other (non) T cells, in particular monocytes, resulting in enhanced co-stimulatory signals. In this regard, the anti-CD3 IgG4P Fwk3-anti-CD3dsscFv also showed activity in this assay (as did the OKT3 control, which is a whole anti-CD3 IgG antibody). These data demonstrate that antibodies of the invention that bind to both CD3 and CD28 are functional activators of human CD4 T cells in a mixed PBMC assay, and are essentially capable of performing the function of a bispecific antibody.

In addition, CD8 T cell activation was also measured in the same assay. As described previously, all positive and negative controls functioned as expected. As can be seen from FIG. 24 (C) and (D), CD69 induction on CD8 T cells for the tested constructs was comparable between donors. Antibodies of the invention that bind CD3 and CD28 (either in Fab or IgG4P format) were the most potent activators of CD8 T cells. Anti-CD3 IgG4P Fwk3 anti-CD3dsscFv also showed activity in this assay (as did the OKT3 control, which is a whole anti-CD3 IgG antibody). These data demonstrate that antibodies of the invention that bind both CD3 and CD28 are functional activators of human CD8 T cells in a mixed PBMC assay and are essentially capable of performing the function of a bispecific antibody.

The results show that the antibodies of the invention have the functionality of both a scaffold antibody (eg, anti-CD28 Fab or IgG4P) and an insert polypeptide, ie. a second antibody (eg, anti-CD3 dsscFv) when the insert polypeptide is present in the Fwk3 region of the framework antibody.

"Containing" in the context of the present description means "including".

When technically possible, embodiments of the invention can be combined. Embodiments are described herein as including certain features/elements. The invention also extends to individual embodiments consisting or essentially consisting of the indicated features/elements.

Of course, it will be understood that the present invention has been described by way of example, it is understood that the examples are not limiting in any way, and modifications may be made to the scope of the claims appended hereinafter. The preferred features of each embodiment of the invention are the same as for other embodiments, mutatis mutandis. All publications, including, but not limited to, patents and patent applications cited in the present description, are incorporated herein by reference, as if it was specifically and individually indicated that each individual publication is included by reference.

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SEQUENCE LIST

<110> YUSB Biopharma SRL

<120> ANTIBODIES CONTAINING POLYPEPTIDE INTEGRATED TO FRAME REGION 3

<130> PF0155-WO

<150>GB1811368.8

<151> 2018-07-11

<160> 144

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Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

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Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

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Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

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Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

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Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

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Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu

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Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

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Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

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Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

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Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

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<213> Artificial sequence

<220>

<223> Flexible linker

<400> 24

Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr

1 5 10 15

Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser His Tyr

20 25

<210> 25

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 25

Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr

1 5 10

<210> 26

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 26

Ala Thr Thr Thr Gly Ser

15

<210> 27

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 27

Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Ser Pro Pro Ser Lys Glu

1 5 10 15

Ser His Lys Ser Pro

20

<210> 28

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 28

Gly Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

1 5 10 15

<210> 29

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 29

Gly Gly Gly Gly Ile Ala Pro Ser Met Val Gly Gly Gly Gly Ser

1 5 10 15

<210> 30

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 30

Gly Gly Gly Gly Lys Val Glu Gly Ala Gly Gly Gly Gly Gly Ser

1 5 10 15

<210> 31

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 31

Gly Gly Gly Gly Ser Met Lys Ser His Asp Gly Gly Gly Gly Ser

1 5 10 15

<210> 32

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 32

Gly Gly Gly Gly Asn Leu Ile Thr Ile Val Gly Gly Gly Gly Ser

1 5 10 15

<210> 33

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 33

Gly Gly Gly Gly Val Val Pro Ser Leu Pro Gly Gly Gly Gly Ser

1 5 10 15

<210> 34

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 34

Gly Gly Glu Lys Ser Ile Pro Gly Gly Gly Gly Ser

1 5 10

<210> 35

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 35

Arg Pro Leu Ser Tyr Arg Pro Pro Phe Pro Phe Gly Phe Pro Ser Val

1 5 10 15

ArgPro

<210> 36

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 36

Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser Pro Ser Leu

1 5 10 15

Thr Thr

<210> 37

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 37

Thr Pro Ser His Leu Ser His Ile Leu Pro Ser Phe Gly Leu Pro Thr

1 5 10 15

Glu Asn

<210> 38

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 38

Arg Pro Val Ser Pro Phe Thr Phe Pro Arg Leu Ser Asn Ser Trp Leu

1 5 10 15

ProAla

<210> 39

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 39

Ser Pro Ala Ala His Phe Pro Arg Ser Ile Pro Arg Pro Gly Pro Ile

1 5 10 15

Arg Thr

<210> 40

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 40

Ala Pro Gly Pro Ser Ala Pro Ser His Arg Ser Leu Pro Ser Arg Ala

1 5 10 15

Phe Gly

<210> 41

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 41

Pro Arg Asn Ser Ile His Phe Leu His Pro Leu Leu Val Ala Pro Leu

1 5 10 15

Gly Ala

<210> 42

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 42

Met Pro Ser Leu Ser Gly Val Leu Gln Val Arg Tyr Leu Ser Pro Pro

1 5 10 15

Asp Leu

<210> 43

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 43

Ser Pro Gln Tyr Pro Ser Pro Leu Thr Leu Thr Leu Pro Pro His Pro

1 5 10 15

Ser Leu

<210> 44

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 44

Asn Pro Ser Leu Asn Pro Ser Tyr Leu His Arg Ala Pro Ser Arg

1 5 10 15

Ile Ser

<210> 45

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 45

Leu Pro Trp Arg Thr Ser Leu Leu Pro Ser Leu Pro Leu Arg Arg Arg

1 5 10 15

Pro

<210> 46

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 46

Pro Pro Leu Phe Ala Lys Gly Pro Val Gly Leu Leu Ser Arg Ser Phe

1 5 10 15

Pro Pro

<210> 47

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 47

Val Pro Pro Ala Pro Val Val Ser Leu Arg Ser Ala His Ala Arg Pro

1 5 10 15

Pro Tyr

<210> 48

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 48

Leu Arg Pro Thr Pro Pro Arg Val Arg Ser Tyr Thr Cys Cys Pro Thr

1 5 10 15

Pro

<210> 49

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 49

Pro Asn Val Ala His Val Leu Pro Leu Leu Thr Val Pro Trp Asp Asn

1 5 10 15

Leu Arg

<210> 50

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 50

Cys Asn Pro Leu Leu Pro Leu Cys Ala Arg Ser Pro Ala Val Arg Thr

1 5 10 15

PhePro

<210> 51

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Rigid linker

<400> 51

Gly Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala

1 5 10

<210> 52

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Rigid linker

<400> 52

Pro Pro Pro Pro

1

<210> 53

<211> 112

<212> PROTEIN

<213> Homo sapiens

<400> 53

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

<210> 54

<211> 352

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 IL-15

<400> 54

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Lys Tyr Gln Ser Gly Gly Ser

65 70 75 80

Gly Met Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

85 90 95

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

100 105 110

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

115 120 125

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

130 135 140

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

145 150 155 160

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

165 170 175

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

180 185 190

Phe Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Lys Asn Thr Val Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

245 250 255

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

260 265 270

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

275 280 285

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

290 295 300

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

305 310 315 320

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

325 330 335

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

340 345 350

<210> 55

<211> 133

<212> PROTEIN

<213> Homo sapiens

<400> 55

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 56

<211> 368

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 IL-2

<400> 56

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Ser Gly Gly

65 70 75 80

Gly Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu

85 90 95

Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn

100 105 110

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

115 120 125

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

130 135 140

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe

145 150 155 160

His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu

165 170 175

Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu

180 185 190

Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln

195 200 205

Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Lys Asn Thr Val Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

225 230 235 240

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

245 250 255

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

260 265 270

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

275 280 285

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

290 295 300

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

305 310 315 320

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

325 330 335

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

340 345 350

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

355 360 365

<210> 57

<211> 178

<212> PROTEIN

<213> Homo sapiens

<400> 57

Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu

1 5 10 15

Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu

20 25 30

Gln Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro

35 40 45

Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro

50 55 60

Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser

65 70 75 80

Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu

85 90 95

Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu

100 105 110

Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu

115 120 125

Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe

130 135 140

Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His

145 150 155 160

Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala

165 170 175

GlnPro

<210> 58

<211> 412

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 G-CSF

<400> 58

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Ala

65 70 75 80

Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys

85 90 95

Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln

100 105 110

Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu

115 120 125

Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu

130 135 140

Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln

145 150 155 160

Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu

165 170 175

Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp

180 185 190

Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly

195 200 205

Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala

210 215 220

Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu

225 230 235 240

Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln

245 250 255

Pro Ser Gly Gly Gly Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser

260 265 270

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro

275 280 285

Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu

290 295 300

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

305 310 315 320

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

325 330 335

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

340 345 350

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

355 360 365

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

370 375 380

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

385 390 395 400

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

405 410

<210> 59

<211> 127

<212> PROTEIN

<213> Homo sapiens

<400> 59

Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val

1 5 10 15

Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr

20 25 30

Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp

35 40 45

Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln

50 55 60

Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met

65 70 75 80

Ala Ser His Tyr Lys Gln His Cys Pro Thr Pro Glu Thr Ser Cys

85 90 95

Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp

100 105 110

Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

115 120 125

<210> 60

<211> 362

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 GM-CSF

<400> 60

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Ser Gly Gly

65 70 75 80

Gly Gly Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu

85 90 95

His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg

100 105 110

Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met

115 120 125

Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr

130 135 140

Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr

145 150 155 160

Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr

165 170 175

Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu

180 185 190

Lys Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln

195 200 205

Glu Gly Gly Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg

210 215 220

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly Tyr

225 230 235 240

Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr

245 250 255

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

260 265 270

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

275 280 285

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

290 295 300

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

305 310 315 320

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

325 330 335

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

340 345 350

Val Asp Lys Lys Val Glu Pro Lys Ser Cys

355 360

<210> 61

<211> 359

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC with GM-CSF at the C-terminus of CH1

<400> 61

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ala Pro Ala Arg Ser Pro Ser Pro

225 230 235 240

Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg

245 250 255

Leu Leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val

260 265 270

Glu Val Ile Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln

275 280 285

Thr Arg Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys

290 295 300

Leu Lys Gly Pro Leu Thr Met Met Ala Ser His Tyr Lys Gln His Cys

305 310 315 320

Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu

325 330 335

Ser Phe Lys Glu Asn Leu Lys Asp Phe Leu Leu Val Ile Pro Phe Asp

340 345 350

Cys Trp Glu Pro Val Gln Glu

355

<210> 62

<211> 124

<212> PROTEIN

<213> Animals of the murine family

<400> 62

Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His Val

1 5 10 15

Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val Thr

20 25 30

Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys Lys

35 40 45

Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu Arg

50 55 60

Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser Tyr

65 70 75 80

Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr Gln

85 90 95

Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu Thr

100 105 110

Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys

115 120

<210> 63

<211> 359

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 mGM-CSF

<400> 63

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Ser Gly Gly

65 70 75 80

Gly Gly Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys

85 90 95

His Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro

100 105 110

Val Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe

115 120 125

Lys Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly

130 135 140

Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala

145 150 155 160

Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu

165 170 175

Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe

180 185 190

Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys Gly Gly

195 200 205

Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

210 215 220

Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala

225 230 235 240

Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250 255

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

260 265 270

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

275 280 285

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

290 295 300

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

305 310 315 320

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

325 330 335

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

340 345 350

Lys Val Glu Pro Lys Ser Cys

355

<210> 64

<211> 356

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC with mGM-CSF at the C-terminus of CH1

<400> 64

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ala Pro Thr Arg Ser Pro Ile Thr

225 230 235 240

Val Thr Arg Pro Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn

245 250 255

Leu Leu Asp Asp Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val

260 265 270

Ser Asn Glu Phe Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu

275 280 285

Lys Ile Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly

290 295 300

Ala Leu Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr

305 310 315 320

Pro Glu Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile

325 330 335

Asp Ser Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys

340 345 350

Pro Gly Glyn Lys

355

<210> 65

<211> 166

<212> PROTEIN

<213> Homo sapiens

<400> 65

Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

1 5 10 15

Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

20 25 30

Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu

65 70 75 80

Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp

85 90 95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

100 105 110

Gly Ala Gln Lys Glu Ala Ile Ser Pro Asp Ala Ala Ser Ala Ala

115 120 125

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

130 135 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala

145 150 155 160

Cys Arg Thr Gly Asp Arg

165

<210> 66

<211> 401

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 EPO

<400> 66

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Ser Gly Gly

65 70 75 80

Gly Gly Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg

85 90 95

Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala

100 105 110

Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val

115 120 125

Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu

130 135 140

Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln

145 150 155 160

Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His

165 170 175

Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg

180 185 190

Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Asp Ala Ala Ser

195 200 205

Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe

210 215 220

Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly

225 230 235 240

Glu Ala Cys Arg Thr Gly Asp Arg Gly Gly Gly Lys Asn Thr Val Tyr

245 250 255

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

260 265 270

Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp

275 280 285

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

290 295 300

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

305 310 315 320

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

325 330 335

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

340 345 350

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

355 360 365

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

370 375 380

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

385 390 395 400

Cys

<210> 67

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VHH6

<400> 67

Asp Val Gln Phe Val Glu Ser Gly Gly Gly Ser Val His Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Asn Cys Ala Thr Ser Gly Tyr Ile Tyr Ser Thr Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala His Ile Tyr Thr Asn Ser Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Arg Pro Ser Ile Arg Cys Ala Ser Phe Ser Ala Thr Glu Tyr

100 105 110

Lys Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 68

<211> 369

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 VHH6

<400> 68

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Asp Val Gln Phe Val Glu Ser Gly Gly Gly Ser Val

85 90 95

His Ala Gly Gly Ser Leu Arg Leu Asn Cys Ala Thr Ser Gly Tyr Ile

100 105 110

Tyr Ser Thr Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu

115 120 125

Arg Glu Gly Val Ala His Ile Tyr Thr Asn Ser Gly Arg Thr Tyr Tyr

130 135 140

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys

145 150 155 160

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

165 170 175

Ile Tyr Tyr Cys Ala Ala Arg Pro Ser Ile Arg Cys Ala Ser Phe Ser

180 185 190

Ala Thr Glu Tyr Lys Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser

195 200 205

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr

210 215 220

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

260 265 270

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

275 280 285

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

290 295 300

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

305 310 315 320

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

325 330 335

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

340 345 350

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

355 360 365

Cys

<210> 69

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VHH15

<400> 69

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ala Ser Gly Tyr Thr Gly Cys Thr

20 25 30

Tyr Asp Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ser Gly Ile Asp Ser Asp Gly Arg Ala Thr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Ser Asn Ala Lys Ile Ala Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Leu Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Asn Leu Gln Cys Leu Arg Tyr Pro Gly Glu Tyr Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 70

<211> 363

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 VHH15

<400> 70

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val

85 90 95

Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ala Ser Gly Tyr

100 105 110

Thr Gly Cys Thr Tyr Asp Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys

115 120 125

Glu Arg Glu Phe Val Ser Gly Ile Asp Ser Asp Gly Arg Ala Thr Tyr

130 135 140

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Ser Asn Ala Lys

145 150 155 160

Ile Ala Val Tyr Leu Gln Met Asp Ser Leu Lys Leu Glu Asp Thr Ala

165 170 175

Met Tyr Tyr Cys Asn Leu Gln Cys Leu Arg Tyr Pro Gly Glu Tyr Tyr

180 185 190

Trp Gly Glyn Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly

225 230 235 240

Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

260 265 270

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

275 280 285

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

290 295 300

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

305 310 315 320

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

325 330 335

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

340 345 350

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

355 360

<210> 71

<211> 190

<212> PROTEIN

<213> Homo sapiens

<400> 71

Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu

1 5 10 15

Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr

20 25 30

Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu

35 40 45

Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg

50 55 60

Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu

65 70 75 80

Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile

85 90 95

Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile

100 105 110

Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly

115 120 125

Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys

130 135 140

Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly

145 150 155 160

Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala

165 170 175

Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr

180 185 190

<210> 72

<211> 88

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human sclerostin central domain

<400> 72

Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg

1 5 10 15

Ser Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro

20 25 30

Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro

35 40 45

Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg

50 55 60

Val Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val

65 70 75 80

Arg Leu Val Ala Ser Cys Lys Cys

85

<210> 73

<211> 314

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab CA645 heavy chain with human sclerostin central domain embedded in framework 3, single S linkers

<400> 73

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ser Cys Arg Glu Leu His

65 70 75 80

Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val

85 90 95

Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro

100 105 110

Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe

115 120 125

Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys

130 135 140

Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser

145 150 155 160

Cys Lys Cys Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg

165 170 175

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly Tyr

180 185 190

Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr

195 200 205

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

210 215 220

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

225 230 235 240

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

245 250 255

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

260 265 270

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

275 280 285

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

290 295 300

Val Asp Lys Lys Val Glu Pro Lys Ser Cys

305 310

<210> 74

<211> 333

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab CA645 heavy chain with human sclerostin central domain embedded in framework 3, Gly-Ser linkers (2X G4S)

<400> 74

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp

85 90 95

Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly

100 105 110

Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys

115 120 125

Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr

130 135 140

Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg

145 150 155 160

Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Ser Gly Gly Gly

165 170 175

Gly Ser Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn

180 185 190

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val

195 200 205

Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr

210 215 220

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

225 230 235 240

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

245 250 255

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

260 265 270

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

275 280 285

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

290 295 300

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

305 310 315 320

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

325 330

<210> 75

<211> 323

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab CA645 heavy chain with human sclerostin central domain embedded in framework 3, rigid linkers

<400> 75

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ile Pro Phe Thr Val Cys

65 70 75 80

Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser

85 90 95

Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala

100 105 110

Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser

115 120 125

Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val

130 135 140

Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg

145 150 155 160

Leu Val Ala Ser Cys Lys Cys Glu Tyr His Gly Leu Gln Lys Asn Thr

165 170 175

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

180 185 190

Tyr Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp

195 200 205

Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

210 215 220

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

225 230 235 240

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

245 250 255

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

260 265 270

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

275 280 285

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

290 295 300

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

305 310 315 320

Lys Ser Cys

<210> 76

<211> 425

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab CA645 heavy chain with mature full-length human sclerostin embedded in framework 3, Gly-Ser linkers (1X G4S)

<400> 76

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gln

65 70 75 80

Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu Leu

85 90 95

Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr Met

100 105 110

Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu Thr

115 120 125

Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr

130 135 140

Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu Val

145 150 155 160

Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly

165 170 175

Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro

180 185 190

Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly Glu

195 200 205

Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys

210 215 220

Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly Thr

225 230 235 240

Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg

245 250 255

Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr Ser Gly Gly

260 265 270

Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala

275 280 285

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly Tyr Ser

290 295 300

Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val

305 310 315 320

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

325 330 335

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

340 345 350

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

355 360 365

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

370 375 380

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

385 390 395 400

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

405 410 415

Asp Lys Lys Val Glu Pro Lys Ser Cys

420 425

<210> 77

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 77

Lys Tyr Gln Ser Gly Gly Ser Gly Met Gly

1 5 10

<210> 78

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 78

Ser Gly Gly Ser Tyr Thr Tyr

15

<210> 79

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 79

Gly Gly Gly Ser Gly Gly Gly Gly

15

<210> 80

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 80

Gly Ser Ser Ser Ser Gly Ser

15

<210> 81

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 81

Ser Gly Gly Gly Gly

15

<210> 82

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Rigid linker

<400> 82

Ile Pro Phe Thr Val

15

<210> 83

<211> 347

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain Fab CA497 with human IL-15 embedded in framework 3, Gly-Ser linkers

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr Arg Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

85 90 95

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

100 105 110

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

115 120 125

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

130 135 140

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

145 150 155 160

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

165 170 175

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

180 185 190

Phe Ile Asn Gly Gly Gly Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr Asp Ala Asn

210 215 220

Arg Gln Asn Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

340 345

<210> 84

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain Fab CA497

<400> 84

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Ser Val Ser Ser Ser

20 25 30

Met Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp

85 90 95

Thr Ala Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 85

<211> 346

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab CA240 heavy chain with human IL-15 embedded in framework 3, Gly-Ser linkers

<400> 85

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Met Arg Asn Lys Asn Tyr Gln Tyr Gly Thr Tyr Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Gly Gly Gly

65 70 75 80

Ser Gly Gly Gly Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

85 90 95

Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

100 105 110

Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

115 120 125

Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile

130 135 140

His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser

145 150 155 160

Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu

165 170 175

Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

180 185 190

Gln Met Phe Ile Asn Gly Gly Gly Lys Asn Ser Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu

210 215 220

Ser Tyr Tyr Gly Phe Thr Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

245 250 255

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

260 265 270

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

275 280 285

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

290 295 300

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

305 310 315 320

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

325 330 335

Val Asp Lys Lys Val Glu Pro Lys Ser Cys

340 345

<210> 86

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain Fab CA240

<400> 86

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Ile Ser

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Asn Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 87

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HyHEL5 heavy chain

<400> 87

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr His Glu Arg Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Gly Val Tyr Tyr Cys

85 90 95

Leu His Gly Asn Tyr Asp Phe Asp Gly Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215

<210> 88

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HyHEL5 light chain

<400> 88

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Gly Arg Asn Pro Thr Phe

85 90 95

Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

Arg Gly Glu Cys

210

<210> 89

<211> 127

<212> PROTEIN

<213> Artificial sequence

<220>

<223> D3L11

<400> 89

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Asp Ser Ile Glu

20 25 30

Tyr Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Ala Arg Glu Gly Val

35 40 45

Ala Ala Leu Tyr Thr His Thr Gly Asn Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys Ala Lys Asn Met Ala Tyr

65 70 75 80

Leu Arg Met Asp Ser Val Lys Ser Glu Asp Thr Ala Ile Tyr Thr Cys

85 90 95

Gly Ala Thr Arg Lys Tyr Val Pro Val Arg Phe Ala Leu Asp Gln Ser

100 105 110

Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 90

<211> 360

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HyHEL5 anti-lysozyme light chain with anti-lysozyme VHH D3L11 embedded in framework 3, Gly-Ser linkers

<400> 90

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser

65 70 75 80

Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala

85 90 95

Ala Ser Gly Ser Thr Asp Ser Ile Glu Tyr Met Thr Trp Phe Arg Gln

100 105 110

Ala Pro Gly Lys Ala Arg Glu Gly Val Ala Ala Leu Tyr Thr His Thr

115 120 125

Gly Asn Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

130 135 140

Gln Asp Lys Ala Lys Asn Met Ala Tyr Leu Arg Met Asp Ser Val Lys

145 150 155 160

Ser Glu Asp Thr Ala Ile Tyr Thr Cys Gly Ala Thr Arg Lys Tyr Val

165 170 175

Pro Val Arg Phe Ala Leu Asp Gln Ser Ser Tyr Asp Tyr Trp Gly Gln

180 185 190

Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser

210 215 220

Met Glu Thr Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Gly Arg

225 230 235 240

Asn Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val

245 250 255

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

260 265 270

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

275 280 285

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

290 295 300

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

305 310 315 320

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

325 330 335

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

340 345 350

Lys Ser Phe Asn Arg Gly Glu Cys

355 360

<210> 91

<211> 352

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HyHEL5 anti-lysozyme light chain with VHH15 anti-IL-6 embedded in framework 3, Gly-Ser linkers

<400> 91

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser

65 70 75 80

Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val

85 90 95

Ala Ala Ser Gly Tyr Thr Gly Cys Thr Tyr Asp Met Arg Trp Tyr Arg

100 105 110

Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly Ile Asp Ser Asp

115 120 125

Gly Arg Ala Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

130 135 140

Gln Ser Asn Ala Lys Ile Ala Val Tyr Leu Gln Met Asp Ser Leu Lys

145 150 155 160

Leu Glu Asp Thr Ala Met Tyr Tyr Cys Asn Leu Gln Cys Leu Arg Tyr

165 170 175

Pro Gly Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

180 185 190

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr

195 200 205

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu Asp Ala Ala Thr

210 215 220

Tyr Tyr Cys Gln Gln Trp Gly Arg Asn Pro Thr Phe Gly Gly Gly Thr

225 230 235 240

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

245 250 255

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

260 265 270

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

275 280 285

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

290 295 300

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

305 310 315 320

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

325 330 335

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345 350

<210> 92

<211> 365

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 light chain with anti-lysozyme VHH D3L11 embedded in framework 3, Gly-Ser linkers

<400> 92

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val

65 70 75 80

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

85 90 95

Cys Ala Ala Ser Gly Ser Thr Asp Ser Ile Glu Tyr Met Thr Trp Phe

100 105 110

Arg Gln Ala Pro Gly Lys Ala Arg Glu Gly Val Ala Ala Leu Tyr Thr

115 120 125

His Thr Gly Asn Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr

130 135 140

Ile Ser Gln Asp Lys Ala Lys Asn Met Ala Tyr Leu Arg Met Asp Ser

145 150 155 160

Val Lys Ser Glu Asp Thr Ala Ile Tyr Thr Cys Gly Ala Thr Arg Lys

165 170 175

Tyr Val Pro Val Arg Phe Ala Leu Asp Gln Ser Ser Tyr Asp Tyr Trp

180 185 190

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

195 200 205

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Phe Thr Leu Thr Ile

210 215 220

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly

225 230 235 240

Tyr Ser Ser Ile Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu

245 250 255

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

260 265 270

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

275 280 285

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

290 295 300

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

305 310 315 320

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

325 330 335

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

340 345 350

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

355 360 365

<210> 93

<211> 360

<212> PROTEIN

<213> Artificial sequence

<220>

<223> heavy chain Fab CA645 with human IL-15 embedded in framework 3 with bovine long linker

<400> 93

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Thr Ser Val His Gln Glu

65 70 75 80

Thr Lys Lys Tyr Gln Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys

85 90 95

Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr

100 105 110

Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys

115 120 125

Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser

130 135 140

Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu

145 150 155 160

Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu

165 170 175

Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile

180 185 190

Val Gln Met Phe Ile Asn Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val

195 200 205

Asp Val Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr

225 230 235 240

Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250 255

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

260 265 270

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

275 280 285

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

290 295 300

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

305 310 315 320

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

325 330 335

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

340 345 350

Lys Lys Val Glu Pro Lys Ser Cys

355 360

<210> 94

<211> 350

<212> PROTEIN

<213> Artificial sequence

<220>

<223> heavy chain Fab CA645 with human IL-15 embedded in framework 3 with bovine short linker

<400> 94

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Thr Lys Lys Tyr Gln

65 70 75 80

Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

85 90 95

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

100 105 110

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

115 120 125

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

130 135 140

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

145 150 155 160

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

165 170 175

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

180 185 190

Asn Ser Tyr Thr Tyr Asn Tyr Glu Lys Asn Thr Val Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr

210 215 220

Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

245 250 255

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

260 265 270

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

275 280 285

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

290 295 300

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

305 310 315 320

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

325 330 335

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

340 345 350

<210> 95

<211> 356

<212> PROTEIN

<213> Artificial sequence

<220>

<223> heavy chain Fab CA645 with IL-15 embedded in framework 3 with 2xG4S linker

<400> 95

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile

85 90 95

Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu

100 105 110

Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu

115 120 125

Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His

130 135 140

Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser

145 150 155 160

Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu

165 170 175

Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

180 185 190

Met Phe Ile Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Asn

195 200 205

Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe

225 230 235 240

Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

245 250 255

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

260 265 270

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

275 280 285

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

290 295 300

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

305 310 315 320

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

325 330 335

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

340 345 350

Pro Lys Ser Cys

355

<210> 96

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 96

Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser

1 5 10

<210> 97

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 97

Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val

1 5 10

<210> 98

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 98

Glu Thr Lys Lys Tyr Gln Ser

15

<210> 99

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 99

Ser Tyr Thr Tyr Asn Tyr Glu

15

<210> 100

<211> 188

<212> PROTEIN

<213> Homo sapiens

<400> 100

Asn Cys Trp Leu Arg Gln Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr

1 5 10 15

Lys Thr Glu Leu Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser

20 25 30

Thr Ser Trp Thr Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp

35 40 45

Met Ile Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Pro

50 55 60

Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys Gly Pro

65 70 75 80

Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala Leu Leu

85 90 95

Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr Gln Gly

100 105 110

Arg Cys Lys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser Ser Thr

115 120 125

Cys Val Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys Asn Arg

130 135 140

Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly Asn Asp

145 150 155 160

Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr Cys Leu

165 170 175

Leu Gly Arg Ser Ile Gly Leu Ala Gly Glu Tyr Lys

180 185

<210> 101

<211> 627

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab CA645 heavy chain with two human IL-10 monomers embedded in framework 3

<400> 101

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Ser Asn Cys Trp Leu Arg Gln Ala Lys Asn Gly Arg Cys Gln Val Leu

85 90 95

Tyr Lys Thr Glu Leu Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu

100 105 110

Ser Thr Ser Trp Thr Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys

115 120 125

Trp Met Ile Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu

130 135 140

Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys Gly

145 150 155 160

Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala Leu

165 170 175

Leu Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr Gln

180 185 190

Gly Arg Cys Lys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser Ser

195 200 205

Thr Cys Val Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys Asn

210 215 220

Arg Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly Asn

225 230 235 240

Asp Gly Val Thr Tyr Ser Ser Ser Ala Cys His Leu Arg Lys Ala Thr Cys

245 250 255

Leu Leu Gly Arg Ser Ile Gly Leu Ala Gly Glu Tyr Lys Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Cys Trp Leu

275 280 285

Arg Gln Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr Glu Leu

290 295 300

Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser Thr Ser Trp Thr

305 310 315 320

Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile Phe Asn

325 330 335

Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Pro Arg Cys Val Cys

340 345 350

Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys Gly Pro Val Cys Gly Leu

355 360 365

Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala Leu Leu Lys Ala Arg Cys

370 375 380

Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr Gln Gly Arg Cys Lys Lys

385 390 395 400

Thr Cys Arg Asp Val Phe Cys Pro Gly Ser Ser Thr Cys Val Val Asp

405 410 415

Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys Asn Arg Ile Cys Pro Glu

420 425 430

Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly Asn Asp Gly Val Thr Tyr

435 440 445

Ser Ser Ala Cys His Leu Arg Lys Ala Thr Cys Leu Leu Gly Arg Ser

450 455 460

Ile Gly Leu Ala Tyr Glu Gly Gly Gly Gly Ser Gly Ser Lys Asn Thr

465 470 475 480

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

485 490 495

Tyr Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp

500 505 510

Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

515 520 525

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

530 535 540

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

545 550 555 560

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

565 570 575

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

580 585 590

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

595 600 605

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

610 615 620

Lys Ser Cys

625

<210> 102

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL-domain (gL5) CA645-Cys

<400> 102

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 103

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain (gH5) CA645-Cys

<400> 103

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 104

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Rigid linker

<400> 104

Glu Tyr His Gly Leu Gln

15

<210> 105

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain (gH1) CA645

<400> 105

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Thr Thr Val Tyr Leu Gln Met

65 70 75 80

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr

85 90 95

Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 106

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain (gH37) CA645

<400> 106

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Ala Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 107

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain (gH47) CA645

<400> 107

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Thr Val Pro Gly Tyr Ser Ala Ala Pro Tyr Phe Asp Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 108

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain (gL1) CA645

<400> 108

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Lys

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 109

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain (gL4) CA645

<400> 109

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 110

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL-domain (gL4) CA645-Cys

<400> 110

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

<210> 111

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 111

Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp

1 5 10

<210> 112

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 112

Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10

<210> 113

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 113

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly Asp

1 5 10 15

<210> 114

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 114

Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Glu Asp Asp Glu

20

<210> 115

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 115

Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Gly Arg Ser Val

20

<210> 116

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 116

Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Gly Arg Ser Val Lys

20

<210> 117

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 117

Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp

1 5 10 15

<210> 118

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 118

Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 119

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 119

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp

1 5 10 15

<210> 120

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 120

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp

1 5 10 15

<210> 121

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 121

Arg Leu Met Glu Asp Ile Cys Leu Ala Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 122

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 122

Glu Val Arg Ser Phe Cys Thr Arg Trp Pro Ala Glu Lys Ser Cys Lys

1 5 10 15

Pro Leu Arg Gly

20

<210> 123

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 123

Arg Ala Pro Glu Ser Phe Val Cys Tyr Trp Glu Thr Ile Cys Phe Glu

1 5 10 15

Arg Ser Glu Gln

20

<210> 124

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 124

Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met

1 5 10

<210> 125

<211> 224

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain Fab CA497

<400> 125

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr Arg Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Thr Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 126

<211> 223

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain Fab CA240

<400> 126

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Met Arg Asn Lys Asn Tyr Gln Tyr Gly Thr Tyr Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Ser Tyr Tyr Gly Phe Thr Ser Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 127

<211> 247

<212> PROTEIN

<213> Artificial sequence

<220>

<223> dsscFv (vHvL) CA497

<400> 127

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr Arg Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Thr Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe Ala Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser Pro

130 135 140

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys

145 150 155 160

Ala Ser Glu Ser Val Ser Ser Ser Met Tyr Ser Tyr Met His Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala Ser

180 185 190

Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Ser Trp Thr Ala Pro Arg Thr Phe Gly Cys

225 230 235 240

Gly Thr Lys Val Glu Ile Lys

245

<210> 128

<211> 247

<212> PROTEIN

<213> Artificial sequence

<220>

<223> dsscFv (vLvH) CA497

<400> 128

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Ser Val Ser Ser Ser

20 25 30

Met Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp

85 90 95

Thr Ala Pro Arg Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Val Ile Phe Ser Asp Tyr Tyr Met

145 150 155 160

Ala Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ala Ser

165 170 175

Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr Arg Glu Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln

195 200 205

Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr

210 215 220

Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 129

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 CA497 dsscFv (vHvL)

<400> 129

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

85 90 95

Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile

100 105 110

Phe Ser Asp Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Cys

115 120 125

Leu Glu Trp Val Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr

130 135 140

Arg Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys

145 150 155 160

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala

165 170 175

Val Tyr Tyr Cys Thr Thr Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe

180 185 190

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu

210 215 220

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

225 230 235 240

Ile Thr Cys Lys Ala Ser Glu Ser Val Ser Ser Ser Met Tyr Ser Tyr

245 250 255

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

260 265 270

Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

275 280 285

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

290 295 300

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Thr Ala Pro Arg

305 310 315 320

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

325 330 335

Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

340 345 350

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly

355 360 365

Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

385 390 395 400

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

405 410 415

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

420 425 430

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

435 440 445

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

450 455 460

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

465 470 475 480

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

485 490

<210> 130

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 Fab HC Fwk3 CA497 dsscFv (vLvH)

<400> 130

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

85 90 95

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Ser

100 105 110

Val Ser Ser Ser Met Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro

115 120 125

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

130 135 140

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

145 150 155 160

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

165 170 175

Gln Gln Ser Trp Thr Ala Pro Arg Thr Phe Gly Cys Gly Thr Lys Val

180 185 190

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

195 200 205

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro

210 215 220

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile Phe Ser

225 230 235 240

Asp Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu

245 250 255

Trp Val Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr Arg Glu

260 265 270

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

275 280 285

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

290 295 300

Tyr Cys Thr Thr Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe Ala Tyr

305 310 315 320

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

325 330 335

Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

340 345 350

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly

355 360 365

Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

385 390 395 400

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

405 410 415

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

420 425 430

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

435 440 445

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

450 455 460

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

465 470 475 480

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

485 490

<210> 131

<211> 718

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 IgG4P HC Fwk3 CA497 dsscFv (vHvL)

<400> 131

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

85 90 95

Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile

100 105 110

Phe Ser Asp Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Cys

115 120 125

Leu Glu Trp Val Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr

130 135 140

Arg Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys

145 150 155 160

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala

165 170 175

Val Tyr Tyr Cys Thr Thr Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe

180 185 190

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu

210 215 220

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

225 230 235 240

Ile Thr Cys Lys Ala Ser Glu Ser Val Ser Ser Ser Met Tyr Ser Tyr

245 250 255

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

260 265 270

Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

275 280 285

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

290 295 300

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Thr Ala Pro Arg

305 310 315 320

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

325 330 335

Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

340 345 350

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly

355 360 365

Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

385 390 395 400

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

405 410 415

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

420 425 430

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

435 440 445

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

450 455 460

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

465 470 475 480

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

485 490 495

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

500 505 510

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

515 520 525

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

530 535 540

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

545 550 555 560

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

565 570 575

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

580 585 590

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

595 600 605

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

610 615 620

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

625 630 635 640

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

645 650 655

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

660 665 670

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

675 680 685

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

690 695 700

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 132

<211> 718

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 IgG4P HC Fwk3 CA497 dsscFv (vLvH)

<400> 132

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser Gly

65 70 75 80

Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

85 90 95

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Ser

100 105 110

Val Ser Ser Ser Met Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro

115 120 125

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

130 135 140

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

145 150 155 160

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

165 170 175

Gln Gln Ser Trp Thr Ala Pro Arg Thr Phe Gly Cys Gly Thr Lys Val

180 185 190

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

195 200 205

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro

210 215 220

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ile Phe Ser

225 230 235 240

Asp Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu

245 250 255

Trp Val Ala Ser Ile Asn Phe Asn Ala Asp Ile Ser Tyr Tyr Arg Glu

260 265 270

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

275 280 285

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

290 295 300

Tyr Cys Thr Thr Asp Ala Asn Arg Gln Asn Tyr Asp Trp Phe Ala Tyr

305 310 315 320

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

325 330 335

Gly Gly Gly Gly Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

340 345 350

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro Gly

355 360 365

Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

385 390 395 400

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

405 410 415

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

420 425 430

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

435 440 445

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

450 455 460

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

465 470 475 480

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

485 490 495

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

500 505 510

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

515 520 525

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

530 535 540

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

545 550 555 560

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

565 570 575

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

580 585 590

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

595 600 605

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

610 615 620

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

625 630 635 640

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

645 650 655

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

660 665 670

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

675 680 685

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

690 695 700

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 133

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain against CD3

<400> 133

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 134

<211> 222

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC Fab vs. CD3

<400> 134

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 135

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC IgG4P against CD3

<400> 135

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 136

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain against CD28

<400> 136

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 137

<211> 223

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC Fab vs. CD28

<400> 137

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 138

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC IgG4P against CD28

<400> 138

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 139

<211> 488

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-CD28 Fab HC Fwk3 anti-CD3 dsscFv (vHvL)

<400> 139

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Gly Gly Gly Gly Ser

65 70 75 80

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val

85 90 95

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr

100 105 110

Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys

115 120 125

Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

130 135 140

Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser

145 150 155 160

Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr

165 170 175

Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

180 185 190

Tyr Trp Gly Glyn Gly Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly

195 200 205

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

225 230 235 240

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

245 250 255

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

260 265 270

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

275 280 285

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

290 295 300

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

305 310 315 320

Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg

340 345 350

Ser Asp Asp Thr Ala Val Tyr Phe Cys Thr Arg Ser His Tyr Gly Leu

355 360 365

Asp Trp Asn Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

370 375 380

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

385 390 395 400

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

405 410 415

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

420 425 430

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

435 440 445

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

450 455 460

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

465 470 475 480

Lys Lys Val Glu Pro Lys Ser Cys

485

<210> 140

<211> 487

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-CD3 Fab HC Fwk3 anti-CD3 dsscFv (vHvL)

<400> 140

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser

65 70 75 80

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val

85 90 95

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr

100 105 110

Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys

115 120 125

Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

130 135 140

Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser

145 150 155 160

Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr

165 170 175

Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

180 185 190

Tyr Trp Gly Glyn Gly Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly

195 200 205

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

225 230 235 240

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

245 250 255

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

260 265 270

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

275 280 285

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

290 295 300

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

305 310 315 320

Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg

340 345 350

Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His

355 360 365

Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser

370 375 380

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

385 390 395 400

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

405 410 415

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

420 425 430

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

435 440 445

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

450 455 460

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

465 470 475 480

Lys Val Glu Pro Lys Ser Cys

485

<210> 141

<211> 712

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-CD28 IgG4P HC Fwk3 anti-CD3 dsscFv (vHvL)

<400> 141

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Gly Gly Gly Gly Ser

65 70 75 80

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val

85 90 95

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr

100 105 110

Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys

115 120 125

Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

130 135 140

Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser

145 150 155 160

Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr

165 170 175

Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

180 185 190

Tyr Trp Gly Glyn Gly Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly

195 200 205

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

225 230 235 240

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

245 250 255

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

260 265 270

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

275 280 285

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

290 295 300

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

305 310 315 320

Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg

340 345 350

Ser Asp Asp Thr Ala Val Tyr Phe Cys Thr Arg Ser His Tyr Gly Leu

355 360 365

Asp Trp Asn Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

370 375 380

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

385 390 395 400

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

405 410 415

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

420 425 430

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

435 440 445

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

450 455 460

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

465 470 475 480

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

485 490 495

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

500 505 510

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

515 520 525

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

530 535 540

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

545 550 555 560

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

565 570 575

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

580 585 590

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

595 600 605

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr

610 615 620

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

625 630 635 640

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

645 650 655

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

660 665 670

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

675 680 685

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

690 695 700

Ser Leu Ser Leu Ser Leu Gly Lys

705 710

<210> 142

<211> 711

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-CD3 IgG4P HC Fwk3 anti-CD3 dsscFv (vHvL)

<400> 142

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Gly Gly Gly Gly Ser

65 70 75 80

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val

85 90 95

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr

100 105 110

Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys

115 120 125

Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

130 135 140

Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser

145 150 155 160

Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr

165 170 175

Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

180 185 190

Tyr Trp Gly Glyn Gly Thr Pro Val Thr Val Ser Ser Gly Gly Gly Gly

195 200 205

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

225 230 235 240

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

245 250 255

Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

260 265 270

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

275 280 285

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu

290 295 300

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

305 310 315 320

Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg

340 345 350

Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His

355 360 365

Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser

370 375 380

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

385 390 395 400

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

405 410 415

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

420 425 430

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

435 440 445

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

450 455 460

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

465 470 475 480

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

485 490 495

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

500 505 510

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

515 520 525

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

530 535 540

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

545 550 555 560

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

565 570 575

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

580 585 590

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

595 600 605

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

610 615 620

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

625 630 635 640

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

645 650 655

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

660 665 670

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

675 680 685

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

690 695 700

Leu Ser Leu Ser Leu Gly Lys

705 710

<210> 143

<211> 251

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv (VH-VL) CA645

<400> 143

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu

180 185 190

Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

210 215 220

Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr

225 230 235 240

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

245 250

<210> 144

<211> 251

<212> PROTEIN

<213> Artificial sequence

<220>

<223> dsscFv (VH-VL) CA645

<400> 144

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu

180 185 190

Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

210 215 220

Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr

225 230 235 240

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

245 250

<---

Claims (40)

1. Antibody design to improve the pharmacokinetics of the insert polypeptide, where the antibody construct contains a framework antibody that contains a variable (V) domain and an insert polypeptide, where the insert polypeptide is located in the framework region 3 of the V domain, where the V domain is a VH domain and the framework antibody also contains a VL domain, where the framework antibody is a full-length antibody, Fab, Fab', F(ab')2, VHH or scFv, that binds to serum albumin and contains an albumin-binding domain, and where the insert polypeptide is a polypeptide or second antibody that binds to a different antigen compared to the framework antibody, and where the framework antibody provides an improvement in the half-life of the insert polypeptide in the presence of the insert polypeptide, where the antibody design contains a VL domain containing a CDR-L1 sequence of SEQ ID NO:3; CDR-L2 sequence SEQ ID NO:4; CDR-L3 sequence SEQ ID NO:5; and a VH domain containing a CDR-H1 sequence of SEQ ID NO:8; CDR-H2 with the sequence of SEQ ID NO:9; CDR-H3 with the sequence of SEQ ID NO:10. 2. An antibody construct according to claim 1, wherein the insert polypeptide is located between amino acid residues 73 and 76 of the VH domain. 3. The design of the antibody according to claim 2, where the insert polypeptide is in the VH domain: (i) between amino acid residues 73 and 74; (ii) between amino acid residues 74 and 75; or (iii) between amino acid residues 75 and 76. 4. An antibody construct according to claim 2 or 3, wherein one or more amino acid residues between residues 73 and 76 of the VH domain are replaced by an insert polypeptide. 5. An antibody construct according to any one of claims 1 to 4, further comprising a linker sequence of at least one amino acid connecting the N- and/or C-terminus of the insert polypeptide to the framework region 3. 6. An antibody construct according to claim 5, wherein the linker sequence is at least two, three, four, five, six, seven, or eight or more amino acids at the N-terminus of the insert polypeptide portion. 7. An antibody construct according to claim 5 or 6, wherein the linker sequence is at least two, three, four, five, six, seven, or eight or more amino acids at the C-terminus of the insert polypeptide portion. 8. An antibody construct according to any one of claims 1 to 7 which is a full-length IgG. 9. An antibody construct according to any one of claims 1 to 7 which is a dsscFv. 10. An antibody construct according to any one of claims 1 to 7 which is a Fab. 11. An antibody construct according to any one of the preceding claims that is humanized. 12. An antibody construct according to any one of the preceding claims, wherein the serum albumin is human serum albumin. 13. An antibody construct according to any one of the preceding claims, wherein the scaffold antibody comprises a VL domain comprising SEQ ID NO:2 or SEQ ID NO:102. 14. An antibody construct according to claim 13, wherein the scaffold antibody comprises a light chain comprising SEQ ID NO:1. 15. An antibody construct according to any one of the preceding claims, wherein the scaffold antibody comprises a VH domain comprising SEQ ID NO:7 or SEQ ID NO:103. 16. An antibody construct according to claim 15, wherein the scaffold antibody comprises a heavy chain comprising SEQ ID NO:6. 17. An antibody construct according to any one of the preceding claims, wherein the insert polypeptide is a cytokine. 18. An antibody construct according to claim 17, wherein the cytokine is selected from IL-10, IL-15, IL-2, G-CSF, GM-CSF, and EPO. 19. An antibody construct according to any one of claims 1-16, wherein the insert polypeptide is the second antibody. 20. An antibody construct according to claim 19, wherein the second antibody is a scFv, in particular dsscFv, or a domain antibody such as VH or VL or VHH. 21. An antibody construct according to claim 19 or 20, wherein the second antibody binds to IL-17. 22. An isolated DNA molecule encoding an antibody construct according to any one of claims 1-21. 23. A pair of DNA molecules to obtain an antibody construct according to any one of claims 1-21. 24. A cloning vector for producing an antibody construct according to any one of claims 1-21. 25. A pair of cloning vectors for producing an antibody construct according to any one of claims 1-21. 26. An expression vector encoding an antibody construct according to any one of claims 1-21. 27. A pair of expression vectors encoding an antibody construct according to any one of claims 1-21. 28. A host cell for obtaining an antibody construct according to any one of claims 1 to 21, containing a cloning vector according to claim 24. 29. A host cell for obtaining an antibody construct according to any one of claims 1 to 21, containing a pair of cloning vectors according to claim 25. 30. A host cell for obtaining an antibody construct according to any one of claims 1 to 21, containing an expression vector according to claim 26. 31. A host cell for obtaining an antibody construct according to any one of claims 1 to 21, containing a pair of expression vectors according to claim 27. 32. A method for producing an antibody construct according to any one of claims 1 to 21, comprising culturing a host cell according to any one of claims 28 to 31 and isolating the antibody.

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