RU2774823C2 - Improved tnf-binding agents - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to an immunoglobulin single variable domain (hereinafter – ISVD) specifically binding TNFα. A compound and compositions containing the above-mentioned ISVD are also disclosed.

EFFECT: invention is effective for the treatment of inflammatory bowel disease (IBD), irritable bowel syndrome, Crohn’s disease, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, injury of the digestive tract and/or cancer of the digestive tract.

14 cl, 11 dwg, 16 tbl, 7 ex

Description

Statement of the Invention

The present invention relates to amino acid sequences, compounds and polypeptides that bind tumor necrosis factor alpha ("TNF" or "TNF-alpha"). In particular, the present invention relates to improved immunoglobulin heavy chain single variable domains (also referred to as "ISV" or "ISVD") that bind to tumor necrosis factor alpha, as well as proteins, polypeptides and other constructs, compounds, molecules or chemical components. , which include such ISVDs, collectively referred to as TNF binding agents. Other aspects, embodiments, characteristics, applications, and advantages of the invention will become apparent to those skilled in the art based on the present disclosure.

Prior Art

In this application, the amino acid residues/positions in the variable domain of the immunoglobulin heavy chain are indicated by numbering in accordance with Kabat. For convenience, Figure 1 is a table listing some of the amino acid positions discussed herein and numbering them according to some alternative numbering systems (such as Aho and IMGT. However, it should be noted that, unless explicitly stated otherwise, for the present description and claims Kabat numbering is of primary importance; other numbering systems are provided for reference only).

With respect to CDRs, as is well known in the art, there are many conventional approaches for defining and describing a CDR from a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is most commonly used) and the Chothia definition (which is based on the location structural loops). For example, you can provide a link to the site http://www.bioinf.org.uk/abs/. For the purposes of this specification and claims, even if CDR according to Kabat may be mentioned, CDR is most preferably determined based on the Abm definition (which is based on Oxford Molecular's AbM antibody modeling software), and is considered to be an optimal compromise between the Kabat and Chothia definitions. Again, a link is provided to http://www.bioinf.org.uk/abs/.

Inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC) are chronic, disabling, and progressive diseases. Most non-biological drugs, such as aminosalicylates, steroids, and immunomodulators, provide symptomatic improvement but do not stop the underlying inflammatory process or change the course of the disease. The advent of anti-tumor necrosis factor-α (anti-TNF-α) agents (infliximab, adalimumab, certolizumab pegola) has drastically changed the way IBD is treated, changing how the course of the disease has changed (fewer surgeries, fewer hospitalizations, improved quality of life, getting rid of steroids, higher clinical remission and mucosal healing rate in both CD and UC), as well as patient quality of life and work productivity (see Amiot and Peyrin-Biroulet 2015 Ther Adv Gastroenterol 8:66-82). The most common route of administration of these therapeutic proteins is by injection. Since most of these proteins have a short serum half-life, they must be administered frequently or at high doses to be effective. Systemic use is also associated with an increased risk of infection. Together, this leads to patient non-compliance (see Singh et al. 2008 J Pharm Sci 97:2497-2523).

However, unwanted long-term side effects and opportunistic infections, including tuberculosis and non-Hodgkin's lymphoma, are due to generalized immunosuppression and are the result of repeated systemic injections according to the current monoclonal antibody treatment protocol (Ali et al., 2013; Galloway et al., 2011; Ford & Peyrin-Biroulet, 2013; Kozuch & Hanauer, 2008; Schreiber et al., 2007; Syed et al., 2013).

Oral administration of anti-TNF-α antibodies will avoid some of these side effects.

However, these anti-TNF-α agents are complex proteins. Upon oral delivery, degradation occurs in the acidic and protease-rich environment of the gastrointestinal tract (GIT).

Using decoy proteins to protect a therapeutic antibody, a patient trial using an orally administered bovine colostrum polyclonal antibody to human TNF-alpha (AVX-470) was recently conducted by Avaxia Biologics Inc. in patients with ulcerative colitis. However, the study was terminated.

Therefore, there is a need for new drugs for IBD.

ISVDs (and in particular nanobodies) that can bind TNF and their use are well known in the art, e.g. treatment of diseases and disorders associated and/or mediated by TNF-α or TNF-α signaling, such as inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmune hepatitis, autoimmune mumps, type 1 diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, male infertility, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathies, thyroiditis, and vasculitis.

WO 2006/122786 discloses as SEQ ID NO: 125 a specific anti-TNF-α nanobody designated as NC55TNF_NC7 (PMP6C11). The sequence of this prior art nanobody is shown in Table A below as SEQ ID NO: 58. Table A also shows (as SEQ ID NO: 1) an optimized version of the sequence (also referred to in this application as "Reference A") of this agent, binding TNF, from the prior art, together with its CDR (in accordance with the Kabat and Abm conventions). As can be seen from the alignment in Figure 2, this optimized version of the sequence contains, compared to the prior art sequence SEQ ID NO: 58, the following mutations: Q1E, A14P, Q27F, S29F, P40A, A49S, K73N, Q75K, V78L, D82aN, K83R and Q108L (according to Kabat numbering).

WO 2015/1733256 relates to improved immunoglobulin domains including C-terminal extensions preventing binding to so-called pre-existing antibodies ("PEAs"). WO 2015/1733256 discloses SEQ ID NO: 345 as a specific anti-TNF-α nanobody, also referred to as TNF345 (SEQ ID NO: 59). As can be seen from the alignment in Figure 2, the optimized sequence version of Reference A contains, compared to the prior art sequence SEQ ID NO: 59, the following mutations: V11L and L89V (according to Kabat numbering).

A cocktail of VHH-neutralizing llama monoclonal antibody fragments has recently been proposed as a potential oral therapy (Hussack et al., 2011 J Biol Chem 286, 8961–76.). However, Dumoulin et al. (2002 Protein Sci, 11, 500–15), Harmsen et al. (2006 Appl Microbiol Biotechnol, 72, 544–51) and Hussack et al. (2012 Methods Mol Biol, 911, 417–29) state that single domain fragments of llama antibodies appear to be highly susceptible to proteolytic degradation in the human gastrointestinal system.

WO2007/025977 describes the treatment of chronic enterocolitis involving the in situ secretion of oral anti-mTNF nanobodies by the applied bacteria L. lactis.

Statement of the Invention

The present invention is directed to the provision of improved TNF binding agents, in particular, improved anti-TNF compounds and polypeptides, more specifically anti-TNF ISVDs, and even more specifically, improved anti-TNF nanobodies. The improved TNF binding agents provided by the invention are also referred to herein as "TNF binding agents of the invention" or "TNF binding agents".

More specifically, the present invention is directed to providing improved TNF binding agents that would be useful in the treatment of diseases of the digestive tract, such as, for example, inflammatory bowel disease (IBD), irritable bowel syndrome, Crohn's disease, ulcerative colitis, mucositis, aphthous stomatitis , celiac disease, gastrointestinal trauma, and gastrointestinal cancer. These TNF binding agents should preferably be stable and suitable for oral administration.

Orally administered TNF binding agents are expected not only to neutralize TNF in the lumen of the digestive tract, but also to gain access to the lamina propria and submucosa, since the intestinal barrier of the digestive tract can be compromised or compromised by severe inflammation and/or ulceration. including periodontal disease, aphthous stomatitis; bacterial, viral, fungal or parasitic infections of the digestive tract; peptic ulcers; ulcers associated with stress or H. pylori infection; damage caused by reflux of the esophagus; inflammatory bowel disease; damage caused by cancer of the digestive tract; food intolerance, including but not limited to celiac disease, or ulcers caused by NSAIDs or other orally or systemically delivered drugs.

Thus, the invention is directed to improved TNF binding agents which are variants of Reference A and which are characterized by reduced binding to interfering factors (generally referred to as "preexisting antibodies" or "PEAs") that may be present in the sera of certain healthy subjects. as well as patients. Reference is made to WO 2012/175741, WO 2013/024059 and also, for example, Holland et al. (J. Clin. Immunol. 2013, 33(7):1192-203), as well as concurrently pending PCT Publication Application WO2015/173325 (No. PCT/EP2015/060643) by Ablynx N.V. filed May 13, 2015 and entitled “ Improved immunoglobulin variable domains".

As further described herein, the TNF binding agents of the invention have the same combination of CDRs (i.e., CDR1, CDR2, and CDR3) as shown in Reference A.

Some preferred, but non-limiting, TNF binding agents of the invention are listed in Figure 3 as SEQ ID NOs 8-41, 62, 63, 64, 65, 66 and 69. Figure 4 shows the sequence alignment of SEQ ID NOs: 8-41 with SEQ ID NO: 1 (Reference A) and SEQ ID NO: 58 (PMP6C11). Binding agents of SEQ ID NOs 22-41, 62-66 and 69 are examples of TNF binding agents of the invention having a C-terminal alanine extension, ie. an alanine residue at the C-terminus of the ISVD sequence (also sometimes referred to as "position 114"), compared to the C-terminal sequence of VTVSS (SEQ ID NO: 55, as present in Reference A). As described in WO 2012/175741 (but also, for example, in WO 2013/024059 and WO2015/173325), this C-terminal alanine extension can prevent so-called "preexisting antibodies" (presumed to be IgG) from binding to the putative epitope that is located at the C-terminal region of the ISVD. This epitope is believed to include, among other residues, the surface amino acid residues of the C-terminal sequence of VTVSS, as well as the amino acid residue at position 14 (and amino acid residues next/close to it in the amino acid sequence, such as at positions 11, 13 and 15), and may also include the amino acid residue at position 83 (and amino acid residues following/close to it in the amino acid sequence, such as at positions 82, 82a, 82b and 84) and/or the amino acid residue at position 108 (and amino acid residues next/close to it in the amino acid sequence, such as at position 107).

However, while the presence of such a C-terminal alanine (or, in general, a C-terminal extension) can significantly reduce (and in many cases even essentially completely prevent) the binding of "preexisting antibodies" that can be found in the sera of some subjects (both healthy , and patients), it has been found that the sera of some subjects (such as sera from patients with certain immune diseases such as SLE) may contain pre-existing antibodies that can bind to the C-terminal region of the ISVD (when such region is exposed) , even if the ISVD contains such a C-terminal alanine (or, more generally, such a C-terminal extension). Reference is again made to Applicant's pending published PCT application WO 2015/173325, filed May 13, 2015, entitled "Improved Immunoglobulin Variable Domains".

Accordingly, one of the specific objectives of the invention is to provide TNF binding agents that are improved versions of anti-TNF nanobodies, which are referred to here as "Reference A", and which have reduced binding to so-called "preexisting antibodies" and, in particular, of the type described in WO 02015/173325 (i.e. those pre-existing antibodies that can bind to the open C-terminal region of ISVD even in the presence of a C-terminal extension).

Generally, the invention achieves this goal by providing amino acid sequences that are variants of the sequence of SEQ ID NO: 1 that include the following amino acid residues (i.e., mutations compared to the sequence of SEQ ID NO: 1):

- 89T; or

- 89L in combination with 11V; or

- 89L in combination with 110K or 110Q; or

- 89L in combination with 112K or 112Q; or

- 89L in combination with 11V and 110K or 110Q; or

- 89L in combination with 11V and 112K or 112Q; or

- 11V in combination with 110K or 110Q; or

- 11V in combination with 112K or 112Q.

In particular, in the TNF binding agents provided by the present invention:

- the amino acid residue at position 11 is preferably selected from L or V; and

- the amino acid residue at position 89 is preferably selected from T, V or L; and

the amino acid residue at position 110 is preferably selected from T, K or Q; and

the amino acid residue at position 112 is preferably selected from S, K or Q;

so (i) position 89 is represented by T; or (ii) position 89 is represented by L and position 11 is represented by V; or (iii) position 89 is represented by L and position 110 is represented by K or Q; or (iv) position 89 is represented by L and position 112 is represented by K or Q; or (v) position 89 is represented by L and position 11 is represented by V and position 110 is represented by K or Q; or (vi) position 89 is represented by L and position 11 is represented by V and position 112 is represented by K or Q; or (vii) position 11 is represented by V and position 110 is represented by K or Q; or (vii) position 11 is represented by V and position 112 is represented by K or Q.

Of the amino acid sequences provided by the invention, amino acid sequences in which position 89 is represented by T or in which position 11 is represented by V and position 89 is represented by L (if appropriate in combination with a 110K or 110Q mutation and/or a 112K or 112Q mutation, and in particularly in combination with the 110K or 110Q mutation) are particularly preferred. Even more preferred are amino acid sequences in which position 11 is represented by V and position 89 is represented by L, optionally with the 110K or 110Q mutation.

In a particularly preferred embodiment, in the TNF binding agents provided in the present invention, the amino acid residue at position 11 is V, the amino acid residue at position 89 is L, the amino acid residue at position 110 is T, and the amino acid residue at position 112 is yourself S.

In a particularly preferred embodiment, the present invention relates to TNF binding agents, such as immunoglobulin single variable domains (ISVDs), having:

- CDR1 (according to Abm), which is the amino acid sequence of GFTFSTADMG (SEQ ID NO: 5); and

- CDR2 (according to Abm), which is the amino acid sequence RISGIDGTTY (SEQ ID NO: 6); and

- CDR3 (according to Abm), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs, are not taken into account when determining the degree of sequence identity) of at least 85%, preferably at least 90 %, more preferably at least 95%; and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, a total of 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) 11, 89 , 110, or 112 that may be present, and ignoring any C-terminal extension that may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if present, may be present in the framework and/or CDR, but preferably only present in the framework and not in the CDR); and

- the amino acid residue at position 11 is V; and

- the amino acid residue at position 89 is L; and

- the amino acid residue at position 110 is T; and

- the amino acid residue at position 112 is S; and

- the amino acid residue at position 49 is A; and

- the amino acid residue at position 74 is S.

As mentioned, the amino acid sequences provided by the invention described herein can bind (and in particular can specifically bind) to TNF.

The amino acid sequences of the invention preferably have an EC50 value in a cell-based assay using KYM cells described in Example 1, under 3) from WO 04/041862, which is better than 50 nM, more preferably better than 25 nM, such as less than 10 nm.

Table B lists some non-limiting possible combinations of amino acid residues that may be present at positions 11, 89, 110 and 112 in the TNF binding agents of the invention. Combinations that are particularly preferred are in bold and most preferred combinations are indicated in bold/underline.

However, when optimizing the sequences ("sequence optimization") of TNF binding agents in terms of minimizing or eliminating binding sites for preexisting antibodies on the one hand and humanizing on the other hand, various properties of TNF binding agents, including stability, were (notably ) are negatively affected. In particular, melting temperature as a measure of physical stability decreased, production in both the eukaryotic host P. pastoris and prokaryotic host E. coli decreased, and stability in GI fluids decreased.

Instead of "mutating back to original" PEA binding sites and humanizing mutations, thereby compromising sequence optimization, the present inventors surprisingly found that amino acid residues 49 and/or 74 could be changed to satisfy both seemingly mutually exclusive requirements. Tables B-1 and B-2 list some non-limiting possible combinations of amino acid residues that may be present at positions 11, 89, 110, 112, 49 and/or 74 in the TNF binding agents of the invention.

TNF binding agents according to the invention are also as set forth in the description, examples and figures, i.e. they have CDRs that are as described in this application and have a general degree of sequence identity (as described in this application) with the sequence of SEQ ID NO: 1, which is as disclosed in this application, and/or may have a limited the number of "amino acid differences" (as described in this application) with these reference sequences (one of them).

The TNF binding agents of the invention preferably include the following CDRs (according to the Kabat convention):

- CDR1 (according to Kabat), which is the amino acid sequence of TADMG (SEQ ID NO: 2); and

- CDR2 (according to Kabat), which is the amino acid sequence RISGIDGTTYYDEPVKG (SEQ ID NO: 3); and

- CDR3 (according to Kabat), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO:4).

Alternatively, when the CDRs are in accordance with the Abm convention, the TNF binding agents of the invention preferably include the following CDRs:

- CDR1 (according to Abm), which is the amino acid sequence of GFTFSTADMG (SEQ ID NO:5); and

- CDR2 (according to Abm), which is the amino acid sequence RISGIDGTTY (SEQ ID NO:6); and

- CDR3 (according to Abm), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO:4).

The TNF binding agent of the invention preferably also has:

- degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs, are not taken into account when determining the degree of sequence identity) of at least 85%, preferably at least 90 %, more preferably at least 95%; and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, a total of 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) 11, 89 , 110, or 112 that may be present, and ignoring any C-terminal extension that may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if present, may be present in the framework and/or CDR, but preferably only present in the framework and not in the CDR).

With regard to various aspects and preferred aspects of the TNF binding agents provided by the invention, when they come to the degree of sequence identity with respect to SEQ ID NO: 1 and/or the number and kind of "amino acid differences" that may be present in such a binding agent of the invention (i.e., compared to the sequence of SEQ ID NO: 1), it should be noted that when it is said that (i) the amino acid sequence of the invention has a degree of sequence identity with the sequence of SEQ ID NO: 1 of at least 85%, preferably at least 90%, more preferably at least 95% (wherein CDR, any C-terminal extension that may be present, and mutations at positions 11, 89, 110 and/or 112 required by a particular aspect are not considered for determining degree of sequence identity); and/or when it is said that (ii) the amino acid sequence of the invention has no more than 7, preferably no more than 5, e.g. only 3, 2 or 1 "amino acid differences" with the sequence of SEQ ID NO: 1 (again, not taking into account (taking into account any C-terminal extension that may be present, and not taking into account mutations at positions 11, 89, 110 and/or 112 required by a particular aspect), then it also includes sequences that have no amino acid differences with the SEQ sequence ID NO: 1, except for mutations at positions 11, 89, 110 and/or 112 required by a particular aspect, and any C-terminal extension that may be present.

Thus, in one specific aspect of the invention, the TNF binding agents of the invention may have 100% sequence identity with SEQ ID NO: 1 (including the CDR, but not taking into account the mutation(s) or combination of mutations at positions 11, 49, 74 , 89, 110 and/or 112 disclosed herein and/or any C-terminal extension that may be present), and/or may not have amino acid differences from SEQ ID NO: 1 (i.e., other than a mutation ( mutation)) or a combination of mutations at positions 11, 89, 110 and/or 112 disclosed in this application, and any C-terminal extension that may be present).

When any amino acid differences are present (i.e., in addition to the C-terminal extension and mutations at positions 11, 89, 110 and/or 112, which are required by a specific aspect of the present invention), these amino acid differences may be present in the CDR and/or in frame regions, but preferably they are only present in the frame regions (as defined by the Abm convention, i.e. not in the CDR as defined by the Abm convention), i.e. that the TNF binding agents of the present invention have the same CDRs (defined according to the Abm convention) that are present in SEQ ID NO: 1.

In addition, when the TNF binding agent of the invention according to any aspect of the invention has one or more amino acid differences with the sequence of SEQ ID NO: 1 (other than mutations at positions 11, 89, 110 and/or 112, which are required by a particular aspect ), then some specific but non-limiting examples of such mutations/amino acid differences that may be present (i.e. compared to the sequences of SEQ ID NO: 1) are, for example, E1D, P40A, P40L, P40S (and, in particular P40A), S49A, A74S, L78V, T87A or any suitable combination thereof. In addition, TNF binding agents of the invention may suitably contain (suitable combination of) D60A, E61D and/or P62S mutations, in particular as an ADS motif at positions 60-62 (see SEQ ID NO: 39 for a non-limiting example) . Other examples of mutations are (suitable combination of) one or more suitable "humanizing" substitutions; reference is made, for example, to WO 2009/138519 (or to the prior art referred to in WO 2009/138519) and WO 2008/020079 (or to the prior art referred to in WO 2008/020079), as well as to Tables A- 3-A-8 of WO 2008/020079 (which are lists showing possible humanizing substitutions).

Of the above mutations, the presence of the S49A, A74S and/or L78V mutation (or any suitable combination of any two of them, including all three) is preferred (see SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41 , and 62-63). Figure 5 shows the alignment of SEQ ID NO: 1, SEQ ID NO: 31 and SEQ ID NO 36-41.

In addition, when the TNF binding agents of the invention are present and/or form the N-terminal portion of the polypeptide or compound in which they are present, then they preferably contain a D at position 1 (i.e., an E1D mutation compared to Reference A). Accordingly, in a further aspect, the invention relates to a polypeptide of the invention (which is described hereinafter) which has a TNF binding agent of the invention (which is described below) at its N-terminus, wherein said TNF binding agent of the invention has D in position 1.

Similarly, when the TNF binding agent of the invention is used in a monovalent format, it preferably has both a C-terminal extension X(n) as described herein and a D at position 1.

Some preferred, but non-limiting examples of monovalent TNF binders with a D at position 1 and a C-terminal extension are given as SEQ ID NOS: 40, 39, 36, 64, 69, 37, 38, 41, and 62-63, preferably SEQ ID NOs: 36, 39 and 40, most preferably SEQ ID NOs: 40. In this regard, it should be noted that TNF binding agents with SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41 and 62-63 can also be used in a compound or polypeptide of the invention that is not in a monovalent format. In this case, when the TNF binding sequences of SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41, and 62-63 will preferably have an E instead of a D at their N-terminus (i.e., a D1E mutation, compared to the sequences of SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41 and 62-63) when they are not present at the N-terminal end of the specified polypeptide or compound of the invention (instead, N- the terminal ISVD of said polypeptide or compound of the invention will have a D at position 1; and they will also preferably not contain a C-terminal extension (such as the C-terminal alanine present in SEQ ID NOS: 40, 39, 36, 64, 69, 37, 38, 41 and 62-63) when they are not present at the C-terminus of the compound or polypeptide (instead, preferably the C-terminal ISVD of said polypeptide or compound of the invention will have a C-terminal extension).

With preferred but non-limiting examples, SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, and 62-63 are also examples of TNF binding agents of the invention having additional amino acid differences from SEQ ID NO: 1 , i.e. S49A, A74S and/or L78V. Thus, in a specific aspect, the invention relates to TNF binding agents of the invention (i.e. having mutations at positions 11, 89, 110 and/or 112 as described herein and further as described herein), which at least have a suitable combination of the S49A, A74S and/or L78V mutation, and preferably a suitable combination of any two of these mutations, such as all three of these mutations.

The TNF binding agents of the present invention when they are used in a monovalent format and/or when they are present at the C-terminus and/or form the C-terminus of the protein, polypeptide or other compound or construct in which they are present (or when they are otherwise have an “open” C-terminus on such a protein, polypeptide, or other compound or construct, this generally means that the C-terminus of the ISVD is not associated with or linked to a constant domain (such as the CH1 domain); reference is again made to WO 2012/175741 and WO 2015/1733256) preferably also have a C-terminal extension of formula (X) _n in which n is from 1 to 10, preferably from 1 to 5, for example 1, 2, 3, 4 or 5 (and preferably 1 or 2, eg 1); and each X is a (preferably natural) amino acid residue independently selected from naturally occurring amino acid residues (although in one preferred aspect it does not include any cysteine residues), and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

In accordance with some preferred non-limiting examples of such C-terminal extensions, X _(n) , X and n can be as follows:

- n = 1 and X = Ala;

- n = 2, and each X = Ala;

- n = 3, and each X = Ala;

- n = 2 and at least one X = Ala (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n = 3 and at least one X = Ala (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n = 3 and at least two X = Ala (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n = 1 and X = Gly;

- n = 2 and each X = Gly;

- n = 3 and each X = Gly;

- n = 2 and at least one X = Gly (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n=3 and at least one X=Gly (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n=3 and at least X=Gly (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

- n = 2 and each X = Ala or Gly;

- n = 3 and each X = Ala or Gly;

- n = 3 and at least one X = Ala or Gly (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile); or

- n = 3 and at least two X = Ala or Gly (wherein the remaining amino acid residues (residue) X are independently selected from natural amino acids, but preferably independently selected from Val, Leu and/or Ile);

where aspects (a), (b), (c), (g), (h), (i), (m) and (n) are particularly preferred, aspects in which n = 1 or 2 are preferred, and aspects in which n=1 are particularly preferred.

It should also be noted that preferably each C-terminal extension present in the TNF binding agent of the invention does not contain a (free) cysteine residue (unless said cysteine residue is used or intended for additional functionalization, for example, for pegylation).

Some specific, but non-limiting examples of suitable C-terminal extensions are the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA, or GAG.

When the TNF binding agents of the present invention contain mutations at positions 110 or 112 (optionally in combination with mutations at positions 11 and/or 89 as described herein), the C-terminal amino acid residues from framework 4 (starting from positions 109) may be as follows: (i) if no C-terminal extension is present: VTVKS (SEQ ID NO: 43), VTVQS (SEQ ID NO: 44), VKVSS (SEQ ID NO: 45) or VQVSS (SEQ ID NO: 45) : 46); or (ii) if a C-terminal extension is present: VTVKSX _(n) (SEQ ID NO: 47), VTVQSX(n) (SEQ ID NO: 48), VKVSSX(n) (SEQ ID NO: 49) or VQVSSX _{(n )} (SEQ ID NO: 50), such as VTVKSA (SEQ ID NO: 51), VTVQSA (SEQ ID NO: 52), VKVSSA (SEQ ID NO: 53) or VQVSSA (SEQ ID NO: 54). When the TNF binding agents of the invention do not contain mutations at positions 110 or 112 (but only mutations at positions 11 and/or 89 as described herein), the C-terminal amino acid residues from framework 4 (starting at position 109) are usually are either: (i) when no C-terminal extension is present: VTVSS (SEQ ID NO: 55) (as in SEQ ID NO: 1); or (ii) when a C-terminal extension is present: VTVSSX _(n) (SEQ ID NO: 56), eg VTVSSA (SEQ ID NO: 57). In these C-terminal sequences, X and n are as defined herein for C-terminal extensions.

As can be seen from the alignment in Figure 4 as well as Figure 3, the TNF binding agents of SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41 and 62-63 have both the E1D mutation and C-terminal alanine extension. As mentioned, the presence of a D at position 1 as well as a C-terminal extension makes these TNF binders (and other TNF binders of the invention with a D at position 1 and a C-terminal extension) particularly suitable for use in a monovalent format. (i.e. for the purposes described in this application).

Accordingly, in a further aspect, the invention provides a TNF binding agent (as described below) that has a D at position 1 and a C-terminal extension X(n) (which is preferably a single Ala residue). Said TNF binding agents are preferably (used and/or intended to be used) in a monovalent format. In one specific aspect, said monovalent TNF binding agent is selected from SEQ ID NOS: 40, 39, 36, 64, 69, 37, 38, 62, 63 and 41.

Some preferred, but non-limiting, examples of TNF binding agents of the invention are set forth in SEQ ID NOs: 8-41 and 62-69, and each of these sequences forms another aspect of the invention (as well as proteins, polypeptides, or other compounds or constructs that contain one of these sequences).

Some particularly preferred TNF binding agents of the invention are SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41, and 62-63 (or, as described herein, variants thereof with E at position 1 and/or no C-terminal extension, depending on their intended use in a polypeptide or compound of the invention).

Thus, in a first aspect, the invention relates to a single immunoglobulin variable domain having:

- CDR1 (according to Kabat), which is the amino acid sequence of TADMG (SEQ ID NO: 2); and

- CDR2 (according to Kabat), which is the amino acid sequence RISGIDGTTYYDEPVKG (SEQ ID NO: 3); and

- CDR3 (according to Kabat), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90% , more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present and disregard any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, may be present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

wherein:

- the amino acid residue at position 11 is preferably selected from L or V; and

- the amino acid residue at position 89 is preferably selected from T, V or L; and

the amino acid residue at position 110 is preferably selected from T, K or Q; and

the amino acid residue at position 112 is preferably selected from S, K or Q;

so (i) position 89 is represented by T; or (ii) position 89 is represented by L and position 11 is represented by V; or (iii) position 89 is represented by L and position 110 is represented by K or Q; or (iv) position 89 is represented by L and position 112 is represented by K or Q; or (v) position 89 is represented by L and position 11 is represented by V and position 110 is represented by K or Q; or (vi) position 89 is represented by L and position 11 is represented by V and position 112 is represented by K or Q; or (vii) position 11 is represented by V and position 110 is represented by K or Q; or (vii) position 11 is represented by V and position 112 is represented by K or Q.

In another aspect, the invention relates to a single immunoglobulin variable domain having:

- CDR1 (according to Kabat), which is the amino acid sequence of TADMG (SEQ ID NO: 2); and

- CDR2 (according to Kabat), which is the amino acid sequence RISGIDGTTYYDEPVKG (SEQ ID NO: 3); and

- CDR3 (according to Kabat), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

where the immunoglobulin single variable domain includes the following amino acid residues (i.e. mutations, compared to the amino acid sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 89T; or

- 89L in combination with 11V; or

- 89L in combination with 110K or 110Q; or

- 89L in combination with 112K or 112Q; or

- 89L in combination with 11V and 110K or 110Q; or

- 89L in combination with 11V and 112K or 112Q; or

- 11V in combination with 110K or 110Q; or

- 11V in combination with 112K or 112Q.

In particular, the TNF binding agents of the invention preferably have no more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2, or 1 "amino acid differences" (as defined herein, and disregarding any of of the above mutations at position(s) 11, 89, 110, or 112, which may be present and ignoring any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, may be present in the frameworks and/or CDRs, but preferably only present in the frameworks and not in the CDRs).

In particular, in the TNF binding agents provided by the invention, the amino acid residue at position 11 is V, the amino acid residue at position 89 is L, the amino acid residue at position 110 is T, and the amino acid residue at position 112 is S.

As described herein, some specific but non-limiting examples of such mutations/amino acid differences that may be present (i.e., in a suitable combination) are, for example, E1D, P40A, P40L, P40S (and in particular P40A), S49A, A74S, L78V, T87A, or any suitable combination thereof, as well as, for example, (suitable combination) D60A, E61D and/or P62S mutations (particularly as an ADS motif at positions 60-62) and (suitable combination, see , for example, SEQ ID NO: 39) one or more suitable "humanizing" substitutions. As also mentioned, the presence of the S49A, A74S and/or L78V mutation (or any suitable combination of any two of them, including all three of them) is preferred (as well as the presence of D at position 1 when a TNF binding agent is present and/or forms the N-terminus of a compound or polypeptide of the invention, or is in a monovalent format).

In a preferred aspect, the TNF binding agent of the invention, such as ISVD, includes an alanine at position 49 (A49).

In another preferred aspect, the TNF binding agent of the invention, such as ISVD, comprises serine at position 74 (S74).

In another preferred aspect, the TNF binding agent of the invention, such as ISVD, comprises asparagine at position 73 (N73) and/or lysine at position 75 (K75).

As mentioned, in the invention, amino acid sequences in which position 89 is represented by T or in which position 11 is represented by V and position 89 is represented by L (if appropriate in combination with a 110K or 110Q mutation and/or a 112K or 112Q mutation, and especially in combination with a 110K or 110Q mutation) are particularly preferred. Even more preferred are amino acid sequences in which position 11 is represented by V and position 89 is represented by L, optionally with the 110K or 110Q mutation.

Thus, in one preferred aspect, the invention provides a single immunoglobulin variable domain having:

- CDR1 (according to Kabat), which is the amino acid sequence of TADMG (SEQ ID NO: 2); and

- CDR2 (according to Kabat), which is the amino acid sequence RISGIDGTTYYDEPVKG (SEQ ID NO: 3); and

- CDR3 (according to Kabat), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

wherein:

- the amino acid residue at position 11 is preferably selected from L or V; and

- the amino acid residue at position 89 is represented by T; and

- the amino acid residue at position 110 is preferably suitably selected from T, K or Q (and preferably represented by T); and

the amino acid residue at position 112 is preferably suitably selected from S, K or Q (and preferably represented by S).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain having:

- CDR1 (according to Kabat), which is the amino acid sequence of TADMG (SEQ ID NO: 2); and

- CDR2 (according to Kabat), which is the amino acid sequence RISGIDGTTYYDEPVKG (SEQ ID NO: 3); and

- CDR3 (according to Kabat), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

wherein:

- the amino acid residue at position 11 is represented by V; and

- the amino acid residue at position 89 is represented by L; and

the amino acid residue at position 110 is preferably suitably selected from T, K or Q; and

- the amino acid residue at position 112 is preferably suitably selected from S, K or Q.

In one specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 11V in combination with 89L; or

- 11V in combination with 110K or 110Q;

- 11V in combination with 112K or 112Q;

- 11V in combination with 89L and 110K or 110Q; or

- 11V in combination with 89L and 112K or 112Q;

and have a CDR (according to Kabat) and share a general degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 as described above.

In another specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 89L in combination with 11V; or

- 89L in combination with 110K or 110Q; or

- 89L in combination with 112K or 112Q; or

- 89L in combination with 11V and 110K or 110Q; or

- 89L in combination with 11V and 112K or 112Q;

and have a CDR (according to Kabat), and have a general degree of sequence identity with the amino acid sequence of SEQ ID NO: 1, which is described in this application.

In another specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 110K or 110Q in combination with 11V; or

- 110K or 110Q in combination with 89L; or

- 110K or 110Q in combination with 11V and 89L;

and have a CDR (according to Kabat), and have a general degree of sequence identity with the amino acid sequence of SEQ ID NO: 1, which is described in this application.

In another specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 112K or 112Q in combination with 11V; or

- 112K or 112Q in combination with 89L; or

- 112K or 112Q in combination with 11V and 89L;

and have a CDR (according to Kabat), and have a general degree of sequence identity with the amino acid sequence of SEQ ID NO: 1, which is described in this application.

In another aspect, the TNF binding agents of the invention include a T at position 89 and have a CDR (according to Kabat) and share a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

In another aspect, the TNF binding agents of the invention comprise a T at position 11 and an L at position 89, and have a CDR (according to Kabat), and share a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 as described in this application.

As already mentioned, the TNF binding agents of the invention according to the above aspects are preferably such that they contain a suitable combination of S49A, A74S and/or L78V mutations, and preferably a suitable combination of any two of these mutations, such as all three of these mutations (again, when the TNF binding agent is monovalent or present at the N-terminus of a compound or polypeptide of the invention, an E1D mutation is also preferred).

In another aspect, the invention relates to a single immunoglobulin variable domain having:

- CDR1 (according to Abm), which is the amino acid sequence of GFTFSTADMG (SEQ ID NO: 5); and

- CDR2 (according to Abm), which is the amino acid sequence RISGIDGTTY (SEQ ID NO: 6); and

- CDR3 (according to Abm), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

wherein:

- the amino acid residue at position 11 is preferably selected from L or V; and

- the amino acid residue at position 89 is T, V or L; and

the amino acid residue at position 110 is preferably suitably selected from T, K or Q; and

the amino acid residue at position 112 is preferably suitably selected from S, K or Q;

so (i) position 89 is represented by T; or (ii) position 89 is represented by L and position 11 is represented by V; or (iii) position 89 is represented by L and position 110 is represented by K or Q; or (iv) position 89 is represented by L and position 112 is represented by K or Q; or (v) position 89 is represented by L and position 11 is represented by V and position 110 is represented by K or Q; or (vi) position 89 is represented by L and position 11 is represented by V and position 112 is represented by K or Q; or (vii) position 11 is represented by V and position 110 is represented by K or Q; or (vii) position 11 is represented by V and position 112 is represented by K or Q.

In another aspect, the invention relates to a single immunoglobulin variable domain having:

- CDR1 (according to Abm), which is the amino acid sequence of GFTFSTADMG (SEQ ID NO: 5); and

- CDR2 (according to Abm), which is the amino acid sequence RISGIDGTTY (SEQ ID NO: 6); and

- CDR3 (according to Abm), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

where the immunoglobulin single variable domain includes the following amino acid residues (i.e. mutations, compared to the amino acid sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 89T; or

- 89L in combination with 11V; or

- 89L in combination with 110K or 110Q; or

- 89L in combination with 112K or 112Q; or

- 89L in combination with 11V and 110K or 110Q; or

- 89L in combination with 11V and 112K or 112Q; or

- 11V in combination with 110K or 110Q; or

- 11V in combination with 112K or 112Q.

As already mentioned, when the TNF binding agent of the invention is used in a monovalent format and/or present at the C-terminus of the compound of the invention (as defined herein), the TNF binding agent (and hence the resulting compound of the invention) preferably has a C-terminal extension X(n), wherein the C-terminal extension may be as described herein for TNF binding agents of the invention and/or as described in WO 2012/175741 or WO 2015/173325.

As mentioned in the invention, amino acid sequences in which position 89 is represented by T or in which position 11 is represented by V and position 89 is represented by L (if appropriate in combination with a 110K or 110Q mutation and/or a 112K or 112Q mutation, and especially in combined with the 110K or 110Q mutation) are particularly preferred. Even more preferred are amino acid sequences in which position 11 is represented by V and position 89 is represented by L, optionally with the 110K or 110Q mutation.

Thus, in one preferred aspect, the invention provides a single immunoglobulin variable domain having:

- CDR1 (according to Abm), which is the amino acid sequence of GFTFSTADMG (SEQ ID NO: 5); and

- CDR2 (according to Abm), which is the amino acid sequence RISGIDGTTY (SEQ ID NO: 6); and

- CDR3 (according to Abm), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

wherein:

- the amino acid residue at position 11 is preferably selected from L or V; and

- the amino acid residue at position 89 is represented by T; and

- the amino acid residue at position 110 is preferably suitably selected from T, K or Q (and preferably represented by T); and

the amino acid residue at position 112 is preferably suitably selected from S, K or Q (and preferably represented by S).

In another preferred aspect, the invention relates to a single immunoglobulin variable domain having:

- CDR1 (according to Abm), which is the amino acid sequence of GFTFSTADMG (SEQ ID NO: 5); and

- CDR2 (according to Abm), which is the amino acid sequence RISGIDGTTY (SEQ ID NO: 6); and

- CDR3 (according to Abm), which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4);

and having:

- the degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 (in which any C-terminal extension that may be present, as well as CDRs are not taken into account to determine the degree of sequence identity), at least 85%, preferably at least 90%, more preferably at least 95%;

and/or

- no more than 7, for example, no more than 5, preferably no more than 3, for example, only 3, 2 or 1 "amino acid differences" (as defined in this application, and not taking into account any of the above mutations in position(s) ) 11, 89, 110, or 112 which may be present, and disregarding any C-terminal extension which may be present) with the amino acid sequence of SEQ ID NO: 1 (in which said amino acid differences, if any, are present in the frameworks and/ or CDRs, but preferably present only in the frameworks and not in the CDRs);

and, if necessary, having:

- C-terminal extension (X) _n in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I);

wherein:

- the amino acid residue at position 11 is represented by V; and

- the amino acid residue at position 89 is represented by L; and

the amino acid residue at position 110 is preferably suitably selected from T, K or Q; and

the amino acid residue at position 112 is preferably suitably selected from S, K or Q.

In one specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 11V in combination with 89L; or

- 11V in combination with 110K or 110Q;

- 11V in combination with 112K or 112Q;

- 11V in combination with 89L and 110K or 110Q; or

- 11V in combination with 89L and 112K or 112Q;

and have a CDR (according to Abm) and have a general degree of identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

In another specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 89L in combination with 11V; or

- 89L in combination with 110K or 110Q; or

- 89L in combination with 112K or 112Q; or

- 89L in combination with 11V and 110K or 110Q; or

- 89L in combination with 11V and 112K or 112Q;

and have a CDR (according to Abm) and have a general degree of identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

In another specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 110K or 110Q in combination with 11V; or

- 110K or 110Q in combination with 89L; or

- 110K or 110Q in combination with 11V and 89L;

and have a CDR (according to Abm) and have a general degree of identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

In another specific, but non-limiting aspect, the TNF binding agents of the invention include the following amino acid residues (i.e., mutations, compared to the sequence of SEQ ID NO: 1) at the positions mentioned (numbering according to Kabat):

- 112K or 112Q in combination with 11V; or

- 112K or 112Q in combination with 89L; or

- 112K or 112Q in combination with 11V and 89L;

and have a CDR (according to Abm) and have a general degree of identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

In another aspect, the TNF binding agents of the invention include a T at position 89, and have a CDR (according to Abm) and have a general degree of identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

In another aspect, the TNF binding agents of the invention include V at position 11 and L at position 89, and have a CDR (according to Abm) and have a general degree of identity with the amino acid sequence of SEQ ID NO: 1 as described herein.

As already mentioned, the TNF binding agents of the invention according to the above aspects are preferably such that they contain a suitable combination of S49A, A74S and/or L78V mutations and preferably a suitable combination of any two of these mutations, such as all three of these mutations. (again, when the TNF binding agent is monovalent or present at the N-terminus of a compound or polypeptide of the invention, preferably also by the E1D mutation).

In another specific, but non-limiting aspect, the invention relates to a TNF binding agent as described herein that is in a monovalent format, and in particular to a TNF binding agent as described herein that is in a monovalent format and which has a D at position 1 (and/or an E1D mutation) and a C-terminal X(n) extension as described herein (eg, a C-terminal alanine residue).

In another specific, but non-limiting aspect, the invention relates to a single immunoglobulin variable domain which is or essentially consists of an amino acid sequence selected from one of the following amino acid sequences: SEQ ID NO: 40, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.

In another specific, but non-limiting aspect, the invention relates to a single immunoglobulin variable domain which is or essentially consists of an amino acid sequence selected from one of the following amino acid sequences: SEQ ID NO: 40, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.

For the purposes of the invention, the digestive tract consists of the mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, large intestine, rectum) and anus.

For purposes of the invention, "gastrointestinal tract" or "GIT" means the stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, large intestine, rectum), and anus. The term "gastric digestion" as used herein refers to digestion in the stomach, small intestine and/or colon.

The term "gastric degradation" of an antibody is used herein to refer to the degradation of an ISVD, compound or polypeptide of the invention in the stomach, small intestine, colon by endogenous or exogenous enzymes present in the stomach, small intestine and colon, or due to exposure to acidic conditions at gastric breakdown.

The term "stabilized ISVD", "stable compounds" and "stable polypeptides", as used in this application, is understood to describe an ISVD, compound or polypeptide, respectively, which has been designed in such a way as to make it more resistant to degradation in the digestive tract when local introduction. Compared to an ISVD, compound or polypeptide that has not been processed or engineered according to the invention, a stabilized ISVD, compound or polypeptide that is engineered according to the invention is degraded more slowly or to a lesser extent by gastric degradation, such as degradation by endogenous or exogenous enzymes present in the stomach, small intestine and colon and/or acidic conditions present in the stomach. "Stabilized ISVDs", "stable compounds" and "stabilized polypeptides" are also referred to as "ISVDs with enhanced resistance to GI degradation", "compounds with enhanced resistance to GI degradation" and "polypeptides with enhanced resistance to GI degradation", respectively.

Topical application of an ISVD, compound, or polypeptide in the digestive tract is difficult because the digestive tract degrades and digests topically applied ISVDs, compounds, and polypeptides. In the stomach, low pH and the protease pepsin degrade ingested ISVDs, compounds, and polypeptides. In the small intestine, the enzymes trypsin and chymotrypsin, among others, break down ingested ISVDs, compounds, and polypeptides. In the colon, bacterial proteases degrade ingested ISVDs, compounds, and polypeptides. ISVDs, compounds and polypeptides with improved resistance to gastric degradation would be preferred for topical use in the gastrointestinal tract.

Stabilized ISVDs, compounds and polypeptides can be obtained by mutating one or more amino acid residues that are susceptible to degradation in the stomach into amino acid residues that are resistant to degradation in the stomach. Stabilized ISVDs, compounds and polypeptides can be obtained by mutating multiple amino acid residues that increase resistance to degradation in the stomach.

Therefore, the present invention relates to the identification of amino acid residues conferring stability to the TNF binding agent of the invention and to improving the stability of the TNF binding agent. Preferably, the TNF binding agent is much more resistant to degradation in the stomach under one or more conditions of low pH or activity of one or more proteases such as pepsin, trypsin, chymotrypsin and/or bacterial proteases.

In one embodiment, the invention relates to a method for identifying an amino acid residue that renders an ISVD, compound, or polypeptide resistant to degradation in the stomach, comprising the steps of: (a) degrading the ISVD, compound, or polypeptide into fragments with one or more proteases; and (b) analyzing the fragments of step (a) using suitable means such as, for example, LC-MS; thereby identifying the amino acid residue(s) conferring resistance of the ISVD, compound or polypeptide to degradation in the stomach.

In one embodiment, the invention relates to a method of increasing the resistance of an ISVD, compound or polypeptide to degradation in the stomach, comprising steps (a) and (b) above, followed by: (c) mutation of the amino acid residue(s) conferring resistance to the ISVD compound or polypeptide to degradation in the stomach; and (d) repeating steps (a) and (b) above; whereby the absence of one or more fragments is indicative of improved stability of the ISVD, compound or polypeptide to degradation in the stomach.

In the present description:

- the term "single immunoglobulin variable domain" (also called "ISV" or "ISVD") is commonly used to refer to immunoglobulin variable domains (which can be heavy chain or light chain domains, including VH, VHH or VL domains) that can form a functional antigen binding site without interaction with another variable domain (eg, without the VH/VL interaction required between the VH and VL domains of a conventional 4-chain monoclonal antibody). Examples of ISVDs will be understood by one of skill in the art and include, for example, nanobodies (including VHH, humanized VHH and/or camelized VH such as human camelized VH), IgNARs, domains, (single domain) antibodies (such as dAb's™), which are VH domains or which are derived from a VH domain and (single domain) antibodies (such as dAb's™) which are VL domains or which are derived from a VL domain. Unless explicitly stated otherwise, ISVDs that are based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. Most preferably, unless expressly stated otherwise herein, the ISVD is a nanobody;

- the term "nanobody" is generally defined in WO 2008/020079 or WO 2009/138519, and thus, in a specific aspect, generally means VHH, humanized VHH, or camelized VH (such as camelized human VH) or, as a rule, , optimized for the VHH sequence (such as, for example, optimized for chemical stability and/or solubility, maximally overlapping with known human framework regions, and maximally expressed). It is noted that the terms "nanobody" or "nanobodies" are registered trademarks of Ablynx N.V and may therefore also be referred to as Nanobody® and/or Nanobody®);

- In general, unless otherwise indicated, ISVDs, nanobodies, polypeptides, proteins and other compounds and constructs mentioned in this application are intended for use in the prevention or treatment of diseases or disorders in humans (and/or, if necessary, also in warm-blooded animals and in particular mammals). Thus, in general, ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs described in this application are preferably such that they can be used and/or can be a suitable component, (biological) drug or other pharmaceutical or therapeutic active compound and/or pharmaceutical product or composition. Such a drug, compound, or product is preferably such that it is suitable for administration to a human, for example, for the prevention or treatment of a subject in need of such prevention or treatment, or, for example, as part of a clinical trial. As further described herein, for this purpose, such drug or compound may contain other components, elements, or connecting units other than the ISVDs provided by the invention (which, as also described herein, may be, for example, one or more other therapeutic components and /or one or more other fragments that affect the pharmacokinetic or pharmacodynamic properties of the ISVD-based or nanobodies-based biological substance, such as its half-life). Suitable examples of such additional therapeutic or other moieties will be understood by those skilled in the art and, for example, can typically include any therapeutically active protein, polypeptide, or other binding domain or binding unit, as well as, for example, modifications such as those described on pages 149 to 152 document WO 2009/138159. The ISVD-based biological agent or nanobody-based biological agent is preferably therapeutic or intended for use as a therapeutic agent (which includes prophylaxis and diagnosis), and for this purpose preferably contains at least one ISVD against a therapeutically relevant target (such as, for example, , RANK-L, vWF, IgE, RSV, CXCR4, IL-23 or other interleukins, etc.). For some specific, but non-limiting examples of such ISVD-based biologicals or nanobody-based biologicals, reference is made in Examples 8-18, as well as, for example, various Ablynx NV applications (such as, for example, but not limited to WO 2004/ 062551, WO 2006/122825, WO 2008/020079 and WO 2009/068627), as well as, for example (without limitation), such applications as WO 2006/038027, WO 2006/059108, WO 2007/063308, WO 2007/ 063311, WO 2007/066016 and WO 2007/085814. In addition, as further described herein, the additional component may be an ISVD or nanobody as described herein directed against a protein (human serum) such as (human) serum albumin; and such an ISVD or nanobody may also find therapeutic applications, in particular and/or to extend the half-life of the TNF binding agents described herein. Reference is made to, for example, WO 2004/041865, WO 2006/122787 and WO 2012/175400 which generally describe the use of serum albumin-binding nanobodies to extend half-life. In addition, in the present specification, unless expressly stated otherwise, all terms mentioned herein have the meaning given in WO 2009/138519 (or in the prior art given in WO 2009/138519) or in WO 2008/020079 (or in the prior art cited in WO 2008/020079). In addition, if the method or technique is not specifically described in this application, they can be performed as described in WO 2009/138519 (or in the prior art cited in WO 2009/138519) or WO 2008/020079 (or in the prior art specified in WO 2008/020079). In addition, as described herein, any pharmaceutical product or composition containing any ISVD or compound of the invention may also contain one or more other components known per se for use in pharmaceutical products or compositions (i.e., depending on the intended pharmaceutical form) and/or, for example, one or more other compounds or active substances intended for therapeutic use (ie, to provide a combination product).

In addition, when used in the present description or claims, the following terms have the same meaning as indicated and/or where applicable, may be defined in the manner described on pages 62-75 of WO 2009/138519: "agonist", "antagonist", "inverse agonist", "nonpolar, uncharged amino acid residue", "polar uncharged amino acid residue", "polar, charged amino acid residue", "sequence identity", "exactly the same" and "amino acid difference (when comparing two amino acid sequences), “(in) substantially isolated (form)”, “domain”, “binding domain”, “antigenic determinant”, “epitope”, “against” or “against” (antigen), “specificity”, and "half-life". In addition, the terms "modulate" and "modulate", "interaction site", "specific to", "cross-block", "cross-block" and "cross-block" and "substantially independent of pH" are , as defined (and/or may be defined as described) on pages 74-79 of WO 2010/130832 Ablynx NV. In addition, when referring to a construct, compound, protein, or polypeptide of the invention, terms such as "univalent", "bivalent" (or "polyvalent"), "bispecific" (or "polyspecific"), and "biparatopic" ( or "polyparatopic") may have the meaning given in WO 2009/138519, WO 2010/130832 or WO 2008/020079.

The term "half-life" as used herein in relation to an ISVD, nanobody, ISVD biological agent, nanobody biological agent, or any other amino acid sequence, compound, or polypeptide mentioned herein can be generally defined as described in paragraph (o ) on page 57 of WO 2008/020079 and, as mentioned therein, refers to the time taken to reduce the serum concentration of an amino acid sequence, compound or polypeptide by 50% in vivo, for example, due to degradation of the sequence or compound and/or clearance or sequestration of a sequence or compound through natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined by any known method such as pharmacokinetic analysis. Suitable techniques are known to the person skilled in the art, and may, for example, typically be as described in paragraph (o) on page 57 of WO 2008/020079. As also mentioned in paragraph (o) on page 57 of WO 2008/020079, half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and area under the pharmacokinetic curve (AUC). In this regard, it should be noted that the term "half-life", as used herein specifically, refers to the t1/2-beta half-life or terminal period (in which t1/2-alpha and/or AUC or both may be ignored) . Reference is made, for example, to the experimental section below, as well as to standard references such as Kenneth, A et al: "Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach" (1996). Also mentioned is "Pharmacokinetics", M Gibaldi & D Perron, publ. Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms "increased half-life" or "increased half-life" are also defined in paragraph (o) on page 57 of WO 2008/020079 and specifically refer to an increase in t1/2-beta, or with an increase in t1/2- alpha and/or AUC, or both, or no increase.

Accordingly, in one aspect, the present invention provides a compound described herein, wherein said compound further comprises a whey protein binding component.

In a further aspect, the present invention relates to a compound described herein, wherein said serum protein binding component binds serum albumin.

When the term is not specifically defined, it has the usual meaning in the art, which is clear to a person skilled in the art. For example, reference is made to standard references such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, Genes II, John Wiley & Sons, New York, N.Y., (1985); Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., "Immunology" (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing/Churchill Livingstone, New York (2005), as well as the general art cited in this application.

Also, as already indicated here, the amino acid residues of the nanobody are numbered according to the general numbering for VH presented by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91) applied to camelid VHH domains in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195; or as mentioned here. According to this numbering, nanobody FR1 contains amino acid residues at positions 1-30, nanobody CDR1 contains amino acid residues at positions 31-35, nanobody FR2 contains amino acids at positions 36-49, nanobody CDR2 contains amino acid residues at positions 50-65, nanobody FR3 contains amino acid residues at positions 66-94, nanobody CDR3 contains amino acid residues at positions 95-102, and nanobody FR4 includes amino acid residues at positions 103-113. [In this regard, it should be noted that (as is well known in the art for VH domains and for VHH domains) the total number of amino acid residues in each of the CDRs may vary, and may not correspond to the total number of amino acid residues indicated by Kabat numbering ( that is, one or more positions according to the Kabat numbering cannot be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, in general, Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. However, as a rule, it can be said that according to Kabat numbering and regardless of the number of amino acid residues in the CDR, position 1 according to Kabat numbering corresponds to the beginning of FR1 and vice versa, position 36 according to Kabat numbering corresponds to the beginning of FR2 and vice versa, position 66 in according to Kabat numbering corresponds to the beginning of FR3 and vice versa, and position 103 according to Kabat numbering corresponds to the beginning of FR4 and vice versa].

Alternative methods for numbering amino acid residues of VH domains, which can also be applied similarly to camelid VHH domains and to nanobodies, is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, aspects and figures will be followed by Kabat numbering, in relation to the Riechmann and Muyldermans VHH domains, unless otherwise indicated.

It should also be noted that the figures, any sequence listing, and Experimental/Examples are provided only to further illustrate the invention, and should not be construed or construed as limiting the scope of the invention and/or the appended claims in any way, unless expressly stated otherwise.

The invention also relates to proteins, polypeptides and other constructs, molecules or chemical entities that contain or essentially consist of the TNF binding agents of the invention as described in this application (i.e., which contain or essentially consist of one or more such TNF binding agents such as one, two or three such TNF binding agents); to methods of expression/production of agents that bind TNF, according to the invention and/or to the expression/production of proteins, polypeptides and other constructs, molecules or chemical compounds containing them; to compositions and products (such as pharmaceutical compositions and products) that include TNF binding agents of the invention and/or proteins, polypeptides and other constructs, molecules or chemical compounds containing them; to the nucleotide sequence and nucleic acids that encode the TNF binding agents of the invention and/or encode proteins or polypeptides containing them; and to the use (and in particular therapeutic, prophylactic and diagnostic uses) of the TNF binding agents of the invention and proteins, polypeptides and other constructs, molecules or chemicals that include them.

These and other aspects, embodiments, advantages, applications and uses of the invention will become apparent from the following description.

Accordingly, in a further aspect, the invention relates to proteins (such as fusion proteins), polypeptides, constructs, compounds or other chemical entities that contain or essentially consist of at least one (such as one, two or three) binding agent TNF (also collectively referred to herein as "compounds of the invention", "polypeptides of the invention", "constructs of the invention" or "fusion proteins of the invention"). Therefore, a compound of the invention may be a polypeptide.

Such compounds of the invention may, in addition to one or more TNF binding agents of the invention, further comprise one or more other amino acid sequences, chemical entities, or moieties. These other amino acid sequences, chemical entities, or fragments may provide one or more of the desired properties of the (resultant) compound of the invention and/or may alter the properties of the (resultant) compound of the invention in a desired manner, for example, provide the (resultant) compound of the invention with the desired biological and /or therapeutic activity (for example, to provide the resulting compound of the invention with affinity and preferential activity against another therapeutically relevant target, so that the resulting compound becomes "bispecific" in relation to TNF and this other therapeutically relevant target) to provide the necessary half-life and/ or (otherwise) modify or improve pharmacokinetic and/or pharmacodynamic properties, target a compound of the invention to specific cells, tissues or organs (including cancer cells and cancerous tissues) to provide a cytotoxic effect and/or serve as a detectable label or marker. Some non-limiting examples of such other amino acid sequences, chemical entities, or components are:

one or more suitable linkers (such as a 9GS, 15GS or 35GS linker); and/or

- one or more binding domains or binding units that are directed against a therapeutically relevant target other than TNF (ie, to provide a compound of the invention that is bispecific for both TNF and another therapeutically relevant target); and/or

- one or more binding domains or binding units that provide an increase in half-life (for example, a binding domain or binding unit that can bind to a serum protein, such as serum albumin); and/or

one or more binding domains or binding units that direct the compound of the invention to the desired cell, tissue or organ (eg a cancer cell); and/or

- one or more binding domains or binding units that provide increased specificity against TNF (they can usually bind to TNF, but usually cannot function against TNF on their own); and/or

a binding domain, binding unit, or other chemical entity that allows the compound of the invention to be internalized into a (desired) cell (eg, an anti-EGFR internalizing nanobody as described in WO 2005/044858); and/or

- a component that improves half-life, such as a suitable polyethylene glycol group (i.e. PEGylation), or an amino acid sequence that provides an increased half-life, such as human serum albumin or a suitable fragment (for example, an albumin hybrid) or, for example, a serum albumin binding peptide as described in WO 2008/068280; and/or

- load, such as cytotoxic load; and/or

- a detectable label or marker, such as a radioactive label or a fluorescent label; and/or

- a label that facilitates the immobilization, detection and/or purification of a compound of the invention, such as a HIS or FLAG3 marker; and/or

- a label that can function, such as a GGC or GGGC C-terminal label; and/or

- C-terminal extension X(n), which can be further described here for TNF binding agents of the invention and/or as described in WO 2012/175741 or WO 2015/173325.

Although usually less preferred, it is also not excluded from the scope of the invention that the compounds of the invention may also contain one or more parts or fragments (preferably human) of a conventional antibody (such as an Fc part or a functional fragment thereof) or one or more constant domains) and/or only from the camel antibody heavy chain (such as one or more constant domains).

In a specific aspect, the present invention relates to a construct that contains or essentially consists of an ISVD, as defined in this application, or a compound, as defined in this application, and which additionally contains one or more other groups, residues, fragments or linking units , optionally linked via one or more peptide linkers.

In a further specific aspect, the present invention relates to a construct as defined herein, wherein said one or more other groups, residues, fragments, or binding units are selected from the group consisting of a polyethylene glycol molecule, whey proteins, or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

When the compounds of the invention contain one or more additional binding domains or binding units (for example, an additional, essentially non-functional anti-TNF binding domain or binding unit that provides increased specificity against TNF, a binding domain or binding unit against a therapeutic target other than TNF, a binding domain or binding unit against a target such as human serum albumin that provides an increased half-life, and/or a binding domain or binding unit that targets a compound of the invention to a particular cell, tissue, or organ and/or that allows the compound of the invention to be internalized per cell), these other binding domains or binding units preferably contain one or more ISVDs, and more preferably all are ISVDs. For example, and without limitation, these one or more additional binding domains or binding units can be one or more nanobodies (including VHH, humanized VHH, and/or camelized VHs such as camelized human VHs), a (single domain) antibody that is a VH domain or which is derived from a VH domain, a dAb which is or essentially consists of a VH domain or which is derived from a VH domain, or even a (single) domain antibody or dAb which is or essentially consists of a VL domain. In particular, these one or more binding domains or binding units, if present, may contain one or more nanobodies, and in particular, all are nanobodies.

When a compound of the invention has an ISVD at its C-terminus (wherein the C-terminal ISVD may be the TNF binding agent of the invention or may, for example, be, if present in the compound of the invention, even more substantially a non-functional anti-TNF ISVD that provides increased specificity against TNF, and ISVD against a therapeutic target other than TNF, ISVD against a target such as human serum albumin which provides an increased half-life, or ISVD which targets a compound of the invention to a specific cell, tissue or organ and/or which allows the compound of the invention to be internalized into a cell), then the compound of the invention (i.e., said C-terminal ISVD) preferably has a C-terminal extension X(n), where the C-terminal extension may be as described herein for agents binding TNF according to the invention and/or as described in WO 2012/175741 or WO2015/173325.

When a compound of the invention contains, in addition to one or more TNF binding agents of the invention, any other ISVDs (wherein one or more additional ISVDs may, as mentioned, be an even more substantially non-functional anti-TNF ISVD that provides increased anti-TNF specificity, ISVD against a therapeutic target other than TNF, ISVD against a target such as human serum albumin that provides an increase in half-life, and/or an ISVD that targets a compound of the invention to a specific cell, tissue, or organ, and/or that allows internalization of the compound according to the invention in a cell); and where such additional ISVDs are nanobodies or are ISVDs that essentially consist of and/or are derived from VH sequences, then in accordance with a preferred aspect of the invention, said one or more (and preferably all) additional ISVDs present in the compound according to the invention will contain in their sequence one or more frame mutations that reduce binding to preexisting antibodies. In particular, according to this aspect of the invention, such additional ISVDs may contain (suitable combination of) amino acid residues/mutations at positions 11, 89, 110 and/or 112 that are described in WO 02015/173325 and/or are essentially as described in this application for TNF binding agents of the invention. In one particular aspect, when a compound of the invention has such an ISVD at its C-terminus (i.e., does not have a TNF binding agent of the invention at its C-terminus), then at least said ISVD that is present and/or forms a C-terminus, has such frame mutations that reduce binding to pre-existing antibodies (and the specified C-terminal ISVD preferably also has a C-terminal extension X(n), as described in this application).

As already mentioned, when a compound of the invention is to have an extended half-life (i.e. compared to the monovalent TNF binding agent of the invention), the compound of the invention preferably contains at least one (e.g. one) ISVD (and in particular , nanobody), which provides such an increase in the half-life. Such an ISVD is usually directed against a suitable serum protein, such as transferrin, and in particular against (human) serum albumin. In particular, such an ISVD or nanobody may be a (single) domain antibody or dAb against human serum albumin as described, for example, in EP 2139918, WO 2011/006915, WO 2012/175400, WO 2014/111550 and may, in particular, be a serum albumin-binding nanobody as described in WO 2004/041865, WO 2006/122787, WO 2012/175400 or WO2015/173325. Particularly preferred ISVDs that bind serum albumin are the Alb-1 nanobody (see WO 2006/122787) or humanized variants such as Alb-8 (WO 2006/122787, SEQ ID NO: 62), Alb-23 (WO 2012 /175400, SEQ ID NO: 1) and other humanized (and preferably also sequence-optimized) variants of Alb-1 and/or variants of Alb-8 or Alb-23 (or, as a rule, ISVDs that have essentially the same CDRs , as Alb-1, Alb-8 and Alb-23).

Again, as already mentioned, such an ISVD that binds serum albumin, if present, may contain in its sequence one or more frame mutations that reduce binding to preexisting antibodies. In particular, when such a serum albumin-binding ISVD is a nanobody or (single) domain antibody that essentially consists of and/or is derived from a VH domain, the serum albumin-binding ISVD may contain (suitable combination of) amino acid residues/mutations at positions 11, 89, 110 and/or 112 which are described in WO 02015/173325 and/or which are essentially as described here for the TNF binding agents of the invention. For example, WO 02015/173325 describes a number of Alb-1, Alb-8 and Alb-23 variants that contain amino acid residues/mutations at positions 11, 89, 110 and/or 112 that reduce binding to preexisting antibodies that may be used in the compounds of the invention.

In a specific, but non-limiting, aspect of the invention, the present invention provides a compound of the invention, such as a polypeptide of the invention, comprising, in addition to one or more TNF-binding constituents, for example, ISVDs, at least one serum albumin-binding constituents, such as ISVDs. , binding serum albumin, preferably binding human serum albumin, as described in this application, where the specified ISVD, binding serum albumin, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 hypervariable regions (CDR1-CDR3, respectively ) in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. Preferably said human serum albumin binding ISVD is selected from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G , Alb82-GG, Alb82-GGG, Alb92, or Alb223 (see Table D).

Again, when such a serum albumin-binding ISVD is present at the C-terminus of a compound of the invention, the serum albumin-binding ISVD (and as a result, the compound of the invention) preferably has a C-terminal extension X(n), with the C-terminal extension may be as described herein for TNF binders of the invention and/or as described in WO 2012/175741 or WO 2015/173325. It also preferably has mutations that reduce binding to pre-existing antibodies, such as (suitable combination) amino acid residues/mutations at positions 11, 89, 110 and/or 112 described in WO 2015/173325.

However, as mentioned, other means of increasing the half-life of a compound of the invention (such as PEGylation, hybridization to human albumin or a suitable fragment thereof, or use of a suitable serum albumin binding peptide), although less preferred, are also included within the scope of the invention.

Accordingly, in one embodiment, the present invention relates to a compound as described herein, wherein said whey protein binding component is a non-antibody polypeptide.

In yet another embodiment, the present invention relates to a compound described in this application, additionally containing PEG.

Generally, when a compound of the invention has an extended half-life (e.g., due to the presence of an ISVD that increases half-life, or any other suitable method for increasing half-life), the resulting compound of the invention preferably has a half-life (as defined herein) which is at least 2 times, preferably at least 5 times, for example at least 10 times or more than 20 times, the half-life of the monovalent TNF binding agent of the invention (as measured in a human and/or in a suitable animal model such as mice or cynomolgus monkeys). In particular, the compound of the invention preferably has a half-life (as defined herein) in human subjects of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days.

It will be appreciated from the disclosure that compounds of the invention that are based on one or more ISVDs may have different "formats", for example, are essentially monovalent, divalent, or trivalent; may be monospecific, bispecific, trispecific, etc., and may be biparatopic (as defined herein and, for example, in WO 2009/068625). For example, a compound of the invention may be:

- (essentially) monovalent, that is (essentially) containing a single agent that binds TNF, according to the invention. As already mentioned, when used in a monovalent format, the TNF binding agent of the invention preferably has a C-terminal extension X(n) as described herein. Such a compound of the invention may also have an extended half-life;

- (essentially) divalent or trivalent and monospecific. Such a compound of the invention will contain two or more anti-TNF ISVDs, which may be the same or different, and when they are different, may be directed against the same epitope on TNF or against different epitopes on TNF (in the latter case, to provide biparatopic or polyparatopic compound of the invention). Such a compound of the invention may also have an extended half-life;

- (essentially) divalent, trivalent (or polyvalent) and bispecific or trispecific (or polyspecific). Such a compound of the invention will be directed against TNF and at least one other target. As described herein, said other target may be, for example, another therapeutically relevant target (i.e., other than TNF) to provide a compound of the invention that is bispecific for TNF and said other therapeutic target. Said other target may also be a target that provides an increased half-life (such as human serum albumin) so as to provide a compound of the invention that has an extended half-life. As also mentioned here, such other target may also target a compound of the invention to specific cells, tissues, or organs, or allow the compound of the invention to be internalized within a cell. It is also possible to combine these approaches/ISVD, for example, to provide a compound of the invention that is bispecific for TNF and at least one other therapeutically relevant target and has an increased half-life.

Again, preferably when these compounds of the invention contain one or more ISVDs other than at least one TNF binding agent of the invention, at least one and preferably all of these other ISVDs will contain in their sequence one or more framework mutations that reduce binding to preexisting antibodies (such as, in particular, a combination of amino acid residues/mutations at positions 11, 89, 110 and/or 112 as described herein for TNF binding agents of the invention and/or as described in general in WO2015/173325). Furthermore, when such compounds of the invention have the TNF binding agent of the invention at their C-terminus, said C-terminal TNF binding agent (and, as a result, the compound of the invention) will preferably have a C-terminal extension X (n), as described in this application. Similarly, when such compounds of the invention have a different ISVD at their C-terminus (i.e., not a TNF binding agent of the invention, but, for example, an ISVD that increases half-life), then said C-terminal ISVD (and, as result, a compound of the invention) will preferably have a C-terminal extension X(n) as described herein and/or will contain in its sequence one or more frame mutations that reduce binding to preexisting antibodies (again, as further described in this application and in WO2015/173325).

As one skilled in the art will appreciate, when a compound of the invention is intended for topical application (e.g., on the skin or in the eye), or, for example, it is to have a (localized) therapeutic effect somewhere, e.g., in the gastrointestinal tract (e.g., after oral administration or via a suppository) or in the lungs (for example, after administration by inhalation), or otherwise intended for direct use at its intended site of action (for example, by direct injection), the compound of the present invention generally does not require an extended half-life. In addition, for the treatment of certain acute conditions or indications, it may be preferable not to have an extended half-life. In these cases, the use of a monovalent compound of the invention, or a compound of the invention (containing a TNF binding agent) without prolonging the half-life (eg, a compound of the invention that is divalent or biparatopic with respect to TNF) is preferred.

Some preferred, but non-limiting, examples of such compounds of the invention are shown schematically in Table C-1 below, and each of these forms is another aspect of the invention. Other examples of suitable compounds of the invention without extending the half-life will be apparent to those skilled in the art based on the disclosure of the present application.

As one of skill in the art will understand, when a compound of the invention is for systemic use and/or for the prevention and/or treatment of a chronic disease or disorder, it will generally be preferred that said compound of the invention have an extended half-life (as defined herein) , i.e. compared to the TNF binding agent(s) present in such a compound of the invention. More preferably, such a compound of the invention contains a half-life increasing ISVD, preferably such as an ISVD and in particular a human serum albumin-binding nanobody (as described herein).

Some preferred, but non-limiting, examples of such compounds of the invention are shown schematically in Table C-2 below, each of which is another aspect of the invention. Other examples of suitable compounds of the invention with extended half-life will be apparent to those skilled in the art based on the description disclosed herein. As a rule, for compounds of the invention with increasing half-life, the presence of a C-terminal extension is preferred.

Single immunoglobulin variable domains can be used as building blocks to produce a compound, such as a polypeptide of the invention, which may optionally contain one or more additional building blocks, such as ISVD, against the same or a different epitope on TNF, and /or against one or more other antigens, proteins or targets than TNF; for example, bricks having a therapeutic mode of action.

Generally, compounds, polypeptides, or constructs that contain or essentially consist of a single building block, a single immunoglobulin variable domain, or a single nanobody, will hereinafter be referred to as "univalent" compounds or "univalent" polypeptides or as "univalent constructs", respectively. Compounds, polypeptides, or constructs that contain two or more building blocks or linking units (e.g., ISVDs) will also be referred to in this application as "polyvalent" compounds, polypeptides, or constructs, as well as building blocks/ISVDs present in such compounds, polypeptides, or constructs will also be referred to herein as being in a "polyvalent format". For example, a "bivalent" compound or polypeptide may contain two single immunoglobulin variable domains, optionally linked via a linker sequence; while a "trivalent" compound or polypeptide may contain three single immunoglobulin variable domains, optionally linked via two linker sequences; while a "quadrivalent" compound or polypeptide may contain four single immunoglobulin variable domains, optionally linked via three linker sequences, and so on.

In a polyvalent compound, polypeptide, or construct, two or more ISVDs, such as nanobodies, may be the same or different, and may be directed against the same antigen or antigenic determinant (e.g., against the same part(s) or epitope(s) , or against different parts or epitopes), or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. Compounds, polypeptides, or constructs that contain at least two building blocks (e.g., ISVDs) in which at least one building block is directed against a first antigen (i.e., TNF) and at least one building block is directed against a second antigen (i.e., other than TNF) will also be referred to as "polyspecific" compounds, polypeptides, or constructs, respectively, and structural elements (such as, for example, ISVD) present in such compounds, polypeptides, or constructs, will also be referred to in this application as present in the "polyspecific format". Thus, for example, a "bispecific" compound or polypeptide of the invention is a compound or polypeptide that contains at least one ISVD directed against a first antigen (i.e., TNF) and at least one more ISVD directed against a second antigen (i.e., TNF). is other than TNF), while a "trispecific" compound or polypeptide of the invention is a compound or polypeptide that contains at least one ISVD directed against the first antigen (i.e., TNF), at least one more ISVD directed against a second antigen (ie, different from TNF) and at least one more ISVD directed against a third antigen (ie, different from both TNF and the second antigen); etc.

"Polyparatopic" compounds, "polyparatopic" polypeptides and "polyparatopic" constructs, such as, for example, "biparatopic" compounds, polypeptides or constructs, or "triparatopic" compounds, polypeptides or constructs, contain or essentially consist of two or more building blocks , each with a different paratope.

Accordingly, the ISVDs of the invention that bind TNF may be in substantially isolated form (as defined herein) or they may form part of a compound, polypeptide, or construct that may contain or essentially consist of one or more ISVDs that bind TNF and which may optionally further contain one or more additional amino acid sequences (all of which are optionally linked through one or more suitable linkers). The present invention relates to a compound, polypeptide or construct that contains or essentially consists of at least one ISVD in accordance with the invention, such as one or more ISVDs of the invention (or suitable fragments thereof), binding TNF, as defined in this application .

One or more ISVDs of the invention may be used as a linking unit or building block in such a compound, polypeptide or construct to provide a monovalent, polyvalent or polyparatopic compound, polypeptide or construct of the invention, respectively, all as described herein. Thus, the present invention also relates to a compound or polypeptide that is monovalent, containing or essentially consisting of one monovalent polypeptide or ISVD according to the invention. Thus, the present invention also relates to a compound, polypeptide or construct, which is a polyvalent compound, polyvalent polypeptide or polyvalent construct, respectively, for example, a divalent or trivalent compound, polypeptide or construct, containing or essentially consisting of two or more ISVDs of of the invention (for polyvalent and polyspecific compounds or polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. (J. Biol. Chem., 276: 7346-7350, 2001), as well as, for example, to WO 96/34103, WO 99/23221 and WO 2010/115998).

The invention also relates to a polyvalent compound or polypeptide (also referred to herein as "polyvalent compound(s) of the invention" and "polyvalent polypeptide(s) of the invention", respectively) comprising or (essentially) consisting of at least one ISVD (or suitable fragments thereof) directed against TNF, preferably human TNF, and one additional ISVD.

In one aspect, in its simplest form, a polyvalent compound, polypeptide, or construct of the invention is a divalent compound, polypeptide, or construct of the invention, comprising a first ISVD, such as an anti-TNF nanobody, and an identical second ISVD, such as an anti-TNF nanobody. , wherein said first and second ISVDs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein). In its simplest form, a polyvalent compound, polypeptide, or construct of the invention may be a trivalent compound, polypeptide, or construct of the invention, comprising a first ISVD, such as an anti-TNF nanobody, identical to a second ISVD, such as an anti-TNF nanobody, and identical to a third ISVD, such as a nanobody directed against TNF, wherein said first, second and third ISVD immunoglobulins, such as nanobodies, can optionally be linked through one or more, and in particular two, linker sequences.

In another aspect, a polyvalent compound, polypeptide, or construct of the invention may be a bispecific compound, polypeptide, or construct of the invention comprising a first ISVD, such as a nanobody directed against TNF, and a second ISVD, such as a nanobody directed against a second antigen, wherein said the first and second ISVDs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein); while the polyvalent compound, polypeptide or construct of the invention may also be a trispecific compound, polypeptide or construct of the invention comprising a first ISVD such as a nanobody directed against TNF, a second ISVD such as a nanobody directed against a second antigen, and a third An ISVD, such as a nanobody directed against a third antigen, wherein said first, second and third ISVDs, such as nanobodies, can optionally be linked via one or more, and in particular two, linker sequences. In a particular aspect, a compound, polypeptide, or construct of the invention is a trivalent, bispecific compound, polypeptide, or construct, respectively. A trivalent, bispecific compound, polypeptide or construct of the invention in its simplest form may be a trivalent compound, polypeptide or construct of the invention (as defined herein) containing two identical ISVDs such as nanobodies against TNF and a third ISVD such as a nanobody directed against another antigen, wherein said first, second and third ISVDs, such as nanobodies, can optionally be linked via one or more, and in particular two, linker sequences.

In another aspect, a compound or polypeptide of the invention is a bispecific compound or polypeptide. A bispecific compound, polypeptide, or construct of the invention in its simplest form may be a divalent compound, polypeptide, or construct of the invention (as defined herein) that contains an ISVD, such as a nanobody against TNF, and a second ISVD, such as a nanobody directed against TNF. against another antigen, wherein said first and second ISVDs, such as nanobodies, can optionally be linked via a linker sequence.

In a preferred aspect, the polyvalent compound, polypeptide or construct of the invention comprises or essentially consists of two or more single immunoglobulin variable domains directed against TNF. In one aspect, the invention relates to a compound, polypeptide or construct that contains or essentially consists of at least two ISVDs according to the invention, such as 2, 3 or 4 ISVDs (or suitable fragments thereof) that bind TNF. Two or more ISVDs may optionally be linked via one or more peptide linkers.

In a preferred aspect, a compound, polypeptide, or construct of the invention comprises or essentially consists of at least two ISVDs, wherein said at least two ISVDs may be the same or different, but of which at least one ISVD is directed against TNF, such as an agent binding TNF.

In a particular aspect, a compound, polypeptide, or construct of the invention comprises or essentially consists of at least two ISVDs, wherein the at least two ISVDs are independently selected from the group consisting of SEQ ID NOs: 8-41 and 61-66 and 69.

Relative affinities may depend on the location of the ISVD in the resulting compound, polypeptide, or construct of the invention. It is understood that the order of the ISVD in the compound or polypeptide of the invention (orientation) may be selected according to the needs of a person skilled in the art. The order of the individual ISVDs, as well as whether the compound or polypeptide includes a linker, is a matter of design. Certain orientations, with or without linkers, may provide preferred binding characteristics over other orientations. For example, the order of the first ISVD (eg, ISVD 1) and second ISVD (eg, ISVD 2) in a compound, polypeptide, or construct of the invention may be (N-terminus to C-terminus): (i) ISVD 1 (eg, nanobody 1) - [linker] - ISVD 2 (for example, nanobody 2); or (ii) ISVD 2 (eg nanobody 2) - [linker] - ISVD 1 (eg nanobody 1); (where linker is optional). The invention covers all directions. Compounds, polypeptides, and constructs that contain ISVDs in an orientation that provides the desired binding characteristics can be easily identified by routine screening.

In the compounds or constructs of the invention, such as the polypeptides of the invention, two or more building blocks, such as, for example, ISVD and optionally one or more other groups, drugs, agents, residues, fragments or linking units can be directly linked to each other. with each other (as, for example, described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof. Suitable spacers or linkers for use in multivalent and polyspecific compounds or polypeptides will be apparent to those skilled in the art, and can generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in creating compounds, constructs, proteins or polypeptides for pharmaceutical use.

In one embodiment, the invention provides a compound or polypeptide as defined herein, wherein said ISVDs are directly linked to each other, or linked through a linker. In another embodiment, the invention relates to a compound or polypeptide as defined herein, wherein a first ISVD and/or a second ISVD and/or possibly a third ISVD and/or possibly an ISVD-binding serum albumin are linked through a linker.

Some particularly preferred linkers and spacers include those used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the prior art referred to above, as well as, for example, linkers that are used in the art to create diabodies or ScFv fragments (in this regard, however, it should be noted that although diabodies and ScFv fragments, the linker sequence used must have a length, degree of flexibility, and other properties that allow the respective VH and VL domains to combine to form a complete antigen-binding site, there are no particular restrictions on the length or flexibility of the linker used in the polypeptide of the invention, since each ISVD, such as nanobody, itself forms a complete antigen-binding site).

For example, the linker may be a suitable amino acid sequence, in particular amino acid sequences from 1 to 50, preferably between 1 and 30, eg 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include glycine-serine linkers, e.g. of the (Gly _x Ser _y ) _z type, such as (e.g. (Gly ₄ Ser) ₃ or (Gly ₃ Ser ₂ ) ₃ as described in WO 99/42077, and the GS30, GS15, GS9 and GS7 linkers described in the Ablynx applications referenced herein (see e.g. WO 06/040153 and WO 06/122825) as well as hinge regions such as those of naturally occurring heavy chain antibodies , or similar sequences (as described in WO 94/04678) Preferred linkers are shown in Table E, eg SEQ ID nos: 85-100.

Some other particularly preferred linkers are polyalanine (such as AAA) as well as GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825) linkers.

In one embodiment, the invention relates to a compound defined in this application, where the specified linker is selected from the group consisting of linkers 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS and 40GS.

Other suitable linkers generally contain organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For example, poly(ethylene glycol) components have been used to bind antibody domains, see eg WO 04/081026.

It is within the scope of the invention that the length, degree of flexibility and/or other properties of the linker(s) used (though not critical, as is usually the case for linkers used in ScFv fragments) may have some influence on the properties of the final compound or construct of the invention. such as a polypeptide of the invention, including but not limited to affinity, specificity, or avidity for a chemokine or one or more other antigens. Based on the description, one skilled in the art will be able to determine the optimal linker(s) for use in a particular compound or construct of the invention, such as a polypeptide of the invention, if necessary after a small number of routine experiments.

For example, in polyvalent compounds or polypeptides of the invention that include building blocks such as ISVDs or nanobodies directed against TNF and other targets, the length and flexibility of the linker is preferably such that they allow each building block, such as ISVD, of the present invention, present in the polypeptide, bind to its own target, such as an antigenic determinant on each of the targets. Again, based on the description disclosed herein, one skilled in the art will be able to determine the optimal linker(s) for use in a particular compound or construct of the invention, such as a polypeptide of the invention, if necessary after a small number of routine experiments.

It is also within the scope of the present invention that the linker(s) used provide one or more other beneficial properties or functionality to the compounds or constructs of the invention, such as the polypeptides of the invention, and/or provide one or more sites for derivatization and/or to attach functional groups (eg, as described herein for the ISVD derivatives of the invention). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, while linkers that form or contain small epitopes or tags can be used for detection, identification and/or purification. Again, based on the description, one skilled in the art will be able to determine the optimal linkers for use in a particular compound, polypeptide, or construct of the invention, if necessary, after a small number of routine experiments.

Finally, when two or more linkers are used in compounds or constructs, such as the polypeptides of the invention, these linkers may be the same or different. Again, based on the description disclosed herein, one skilled in the art will be able to determine the optimal linkers for use in a particular compound or construct or polypeptide of the invention, if necessary after a small number of routine experiments.

For a general description of polyvalent and polyspecific compounds and polypeptides containing one or more nanobodies, and their production, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001; Muyldermans, "Reviews in Molecular Biotechnology", 74 (2001), 277-302; and also, for example, WO 1996/34103, WO 1999/23221, WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

The invention also relates to nucleotide sequences or nucleic acids that encode amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds and constructs described in this application. The invention further includes genetic constructs that contain the aforementioned nucleotide sequences or nucleic acids, and one or more elements for known genetic constructs. The genetic construct may be in the form of a plasmid or a vector. Again, these constructs may be generally as described in Ablynx N.V. published patent applications such as, for example, WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/ 068627.

In one aspect, the invention provides a nucleic acid encoding an ISVD, polypeptide, compound or construct according to the invention.

In another aspect, the invention relates to an expression vector containing a nucleic acid according to the invention.

The invention also relates to hosts or host cells that contain such nucleotide sequences or nucleic acids and/or express (or are capable of expressing) amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds and constructs described in this application. Again, such host cells can generally be as described in Ablynx N.V. published patent applications such as, for example, WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

In one aspect, the invention relates to a host or host cell containing a nucleic acid according to the invention, or an expression vector according to the invention.

The invention also relates to a method for obtaining an amino acid sequence, TNF binding agents, polypeptides, fusion protein, compounds or structures, as described in this application, and this method includes culturing or maintaining a host cell, as described in this application, under conditions, at which said host cell produces or expresses an amino acid sequence, TNF binding agents, polypeptides, fusion protein, compounds, or construct as described herein, and optionally further comprising isolating the amino acid sequence, TNF binding agents, polypeptides, fusion protein, compounds or structures obtained in this way. Again, such methods can be carried out as generally described in Ablynx N.V. published patent applications such as, for example, WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

In a particular aspect, the invention relates to a process for the preparation of an ISVD according to the invention, or a compound according to the invention, or a polypeptide according to the invention, said process at least comprising the following steps:

a) expression in a suitable host cell or host organism or other suitable expression system for a nucleic acid sequence as defined herein; if necessary, followed by the next step:

b) isolating and/or purifying an ISVD according to the invention, a compound according to the invention, or a polypeptide according to the invention, respectively.

The invention also relates to a composition that contains at least one amino acid sequence, a TNF binding agent, a polypeptide, a fusion protein, a compound or a construct, as described in this application.

The invention also relates to a pharmaceutical composition that includes at least one amino acid sequence, a TNF binding agent, a polypeptide, a fusion protein, a compound or a construct, as described in this application, and, if necessary, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally contains one or more additional pharmaceutically active polypeptides and/or compounds. Such preparations, carriers, excipients and diluents may generally be as described in Ablynx NV published patent applications such as WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627. .

When the amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs described herein have an extended half-life, they are preferably administered into the blood stream. Thus, they can be administered by any suitable route that allows amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds, or constructs to enter the bloodstream, such as intravenously, by injection or infusion, or by any other suitable route (including oral administration). , subcutaneous administration, intramuscular administration, administration through the skin, intranasal administration, administration through the lungs, etc.), which allows amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs to enter the bloodstream. Suitable modes and routes of administration will be apparent to those skilled in the art, again, for example, from study of Ablynx NV published patent applications such as WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or W02009/068627.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented or treated using an amino acid sequence, a TNF binding agent, a polypeptide, a fusion protein, a compound, or as described herein, wherein the method includes administering to a subject in need thereof a pharmaceutically active amount of an amino acid sequence, a TNF binding agent, a polypeptide, a fusion protein, a compound or construct of the invention and/or a pharmaceutical composition comprising the same. Diseases and disorders that can be prevented or treated using a polypeptide, fusion protein, compound or construct as described herein will be generally the same as diseases and disorders that can be prevented or treated using a therapeutic component that present in a polypeptide, fusion protein, compound or construct of the invention. In particular, the present invention relates to a compound, composition, construct, polypeptide, TNF binding agent, or ISVD as described herein for use as a drug.

In the context of the present invention, the term "prevention and/or treatment" includes not only the prevention and/or treatment of a disease, but also generally includes preventing the onset of the disease, slowing down or preventing the progression of the disease; preventing or slowing down the development of one or more symptoms associated with the disease; reduction and/or alleviation of one or more symptoms associated with the disease; reduction in the severity and/or duration of the disease and/or any associated symptoms; and/or preventing a further increase in the severity of the disease and/or any associated symptoms; prevention, reduction or restoration of any physiological damage caused by the disease; and generally any pharmacological action that is beneficial to the patient being treated. In order to be a suitable therapeutic agent, the composition of the invention is not required to cause a complete cure or eliminate any symptom or manifestation of a disease. In order to be recognized as useful therapeutic agents, it is believed in the relevant art that drugs used as therapeutic agents can reduce the severity of a given pathological condition, but are not required to abolish every manifestation of the disease. Similarly, a prophylactically administered treatment may not necessarily be 100% effective in preventing the onset of the condition to constitute a useful prophylactic agent. Merely reducing the impact of the disease (eg, reducing the number or severity of its symptoms, or increasing the effectiveness of another treatment, or producing another beneficial effect) or reducing the likelihood of the subject developing or worsening the disease is sufficient.

In one embodiment, an indication that a therapeutically effective amount of the composition has been administered to a patient is a sustained improvement, from baseline, in an indicator that reflects the severity of a particular disorder. Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of an ISVD, compound or polypeptide of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients. By "therapeutically effective amount" of an ISVD, compound or polypeptide of the invention is meant the amount of the composition that produces a therapeutic effect in the subject receiving treatment, at a reasonable benefit/risk ratio applicable to any treatment. The therapeutic effect is sufficient to "treat" the patient, as the term is used here.

In one embodiment, the present invention relates to a composition, construct, compound, TNF binding agent, ISVD, or polypeptide as described herein, for use in the treatment of a disease and/or disorder of the digestive tract.

In one embodiment, the present invention relates to a composition, construct, compound, TNF binding agent, ISVD, or polypeptide as described herein, wherein said disease and/or disorder of the digestive tract is inflammatory bowel disease (IBD), irritable bowel syndrome, Crohn's disease, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, gastrointestinal trauma, and gastrointestinal cancer.

Patients with irritable bowel syndrome have altered intestinal permeability despite few or no histological changes being detected in the intestine (Dunlop Am J Gastroenterol, 2006 Jun, 101 (6): 1288-94). Patients with celiac disease have altered intestinal permeability and characteristic damage to the villi of the small intestine, distinguishing this disease from IBD. Inflammatory bowel disease is believed to be the result of a dysregulated immune response initiated by microbe-host interaction. The immune system reacts to non-pathogenic commensal bacteria, causing chronic inflammation. Similarly, in necrotizing enterocolitis, a stressed, underdeveloped immune system generates an inappropriate response to normal intestinal bacteria, causing a potentially fatal form of colitis (Jilling et al., 2006, J Immunol, 177, 3273-82).

The subject to be treated may be any warm-blooded animal, but in particular a mammal, and in particular a human. As will be clear to a person skilled in the art, the subject to be treated will, in particular, be a human suffering from or at risk of developing the diseases and disorders mentioned in this application.

In another embodiment, the invention relates to a method of immunotherapy, and in particular passive immunotherapy, which comprises administering to a subject suffering from or at risk of developing the diseases and disorders mentioned herein, a pharmaceutically active amount of an amino acid sequence, a TNF binding agent, a polypeptide, a fusion protein, compound or construct according to the invention and/or a pharmaceutical composition containing them.

The amino acid sequence, TNF binding agent, polypeptide, fusion protein, compound or construct, and/or compositions containing them are administered in accordance with a treatment regimen that is suitable for the prevention and/or treatment of the disease or disorder to be prevented or treated. The clinician will generally be able to determine the appropriate treatment regimen depending on such factors as the disease or disorder to be prevented or treated; the severity of the disease being treated and/or the severity of its symptoms, the specific amino acid sequence, the TNF binding agent, polypeptide, fusion protein, compound or construct of the invention, the specific route of administration, and the pharmaceutical composition or formulation to be used; age, sex, body weight, diet, general condition of the patient, and similar factors well known to the clinician.

Typically, the treatment regimen will include the administration of one or more amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs of the invention, or one or more compositions containing them, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to be administered may be determined by the clinician, again based on the above factors.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein, and depending on the particular disease or disorder being treated, the efficacy and/or half-life of the particular fusion proteins or constructs to be used, the specific route of administration, and the particular pharmaceutical formulation or composition used, amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs of the invention are typically administered in an amount of 1 g to 0.01 μg per kg of body weight per day, preferably 0.1 g to 0 .1 microgram per kg of body weight per day, for example, about 1, 10, 100, or 1000 micrograms per kg of body weight per day, either continuously (for example, by infusion), or as a single daily dose, or as several divided doses during the day. Usually the clinician can determine the appropriate daily dose, depending on the factors mentioned in this application. It will also be clear that in particular cases the clinician may choose to deviate from these amounts, for example, based on the above factors and his expert judgment. In general, some administration amount recommendations can be derived from the amounts normally used for comparable conventional antibodies or antibody fragments against the same target, administered in substantially the same manner, taking into account, however, differences in affinity/avidity, potency, biodistribution , half-life, and similar factors well known to the skilled person.

Typically, the above method will use a single amino acid sequence, TNF binding agent, polypeptide, fusion protein, compound or construct of the invention. However, it is within the scope of the present invention to use two or more amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs of the invention in combination.

Amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs of the invention may also be used in combination with one or more other pharmaceutically active compounds or active substances, i.e. as a combined treatment regimen, which may or may not result in synergistic effect. Again, the clinician will be able to select such additional compounds or active substances, as well as the appropriate combination treatment regimen, based on the above factors and his or her expert judgment.

In particular, amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs of the invention may be used in combination with other pharmaceutically active compounds or active ingredients that are or may be used in the prevention and/or treatment of diseases and disorders. which can be prevented or treated by amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds or constructs of the invention, and as a result of which a synergistic effect may or may not be achieved.

The efficacy of a treatment regimen used in accordance with the invention may be determined and/or monitored in any manner known per se for the disease or disorder, as will be understood by the clinician. The physician will also be able, as needed and/or on a case-by-case basis, to change or modify a particular treatment regimen in order to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side effects, and/or to achieve an appropriate balance between obtaining the desired therapeutic effect, on the one hand, and the prevention, limitation or reduction of unwanted side effects, on the other hand.

Typically, the treatment regimen will be carried out until the desired therapeutic effect is achieved and/or until the desired therapeutic effect is to be maintained. Again, this can be determined by a doctor.

Because the amino acid sequences, TNF binding agents, polypeptides, fusion proteins, compounds, or constructs of the invention are capable of binding to TNF, they can in particular be used to prevent and/or treat diseases or disorders that can be treated with other biological drugs (e.g., antibodies, such as adalimumab/HUMIRA™ or Infliximab/REMICADE™) that are capable of binding to TNF and/or modulating TNF. Such diseases and disorders will be apparent to the skilled artisan. The TNF binding agents of the invention can in particular be used for the prevention and treatment of the diseases and disorders mentioned in WO 2004/041862 and WO 2006/122786.

As already mentioned, one specific aspect of the invention relates to agents that bind TNF, according to the invention, which are in a monovalent format. These monovalent TNF binding agents of the invention are particularly suitable for topical administration (including application to the skin, gastrointestinal tract or lungs) and/or topical administration (eg skin), oral administration (eg gastrointestinal tract), suppository administration (again, eg, into the gastrointestinal tract), and/or pulmonary administration (eg, by inhalation). Thus, they can be used for the prevention and/or treatment of diseases and disorders of the skin, lungs or gastrointestinal tract, which can be prevented or treated by the application of a TNF inhibitor to the skin, lungs or gastrointestinal tract, respectively (such as inflammatory and/or autoimmune diseases affecting the skin, lungs, or gastrointestinal tract, respectively).

In one embodiment, the present invention relates to a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD is administered topically to the digestive tract.

In one embodiment, the present invention relates to a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD is administered orally in a dosage form, suitable for oral administration in the gastrointestinal tract (GIT).

In one embodiment, the present invention provides a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD is administered in an oral dosage form. administration to the gastrointestinal tract, where the dosage form is selected from tablets, capsules, tablet powders, granules, emulsions, microemulsions, solutions, suspensions, syrups and elixirs.

In one embodiment, the present invention relates to a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD is administered rectally to treat a disease or digestive tract disorders.

In one embodiment, the present invention relates to a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD is administered rectally in a dosage form for rectal administration, preferably selected from suppositories and enemas.

In one embodiment, the present invention also relates to a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD is administered parenterally via subcutaneous injection. , intradermal injection, intravenous injection, intramuscular injection, intralesional injection, or via infusion techniques.

Some specific, but non-limiting, examples of such diseases and disorders are diseases or disorders of the gastrointestinal tract, such as inflammatory bowel disease (IBD), Crohn's disease, irritable bowel syndrome, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, gastrointestinal trauma, and/or cancer. digestive tract. As mentioned, when used for this purpose, the univalent TNF binding agents of the invention preferably have a D (or E1D) mutation at position 1 and a C-terminal extension (such as C-terminal alanine), and TNF binding agents with SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41, 62-63 and 65-66, in particular SEQ ID NOs: 40, 39, 36, 64 and 69, in particular SEQ ID NOs: 40 are preferred examples of TNF binders of the invention which are particularly suitable for this purpose.

Thus, in yet another aspect, the invention provides a TNF binding agent of the invention (as described herein) that is in substantially monovalent format (and which is preferably a D or E1D mutation at position 1 and a C-terminal extension, such as C-terminal alanine) for use in the prevention and treatment of diseases and disorders of the skin, lungs or gastrointestinal tract, and in particular in the prevention and treatment of inflammatory and/or autoimmune diseases affecting the skin, lungs or gastrointestinal tract . Said TNF binding agent is preferably selected from the group consisting of SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41, 62-63 and 65-66, in particular SEQ ID NOs: 40, 39 , 36, 64 and 69, in particular SEQ ID NO: 40.

The invention also relates to a TNF binding agent of the invention (as described herein) which is essentially in a monovalent format (and which is preferably a D or E1D mutation at position 1 and a C-terminal extension such as C-terminal alanine ) for use in the prevention and treatment of diseases or disorders of the gastrointestinal tract, such as inflammatory bowel disease (IBD), Crohn's disease, irritable bowel syndrome, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, trauma of the digestive tract and / or cancer of the digestive tract, in particular , IBD and Crohn's disease. Again, said TNF binding agent is preferably selected from the group consisting of SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41, 62-63 and 65-68, in particular SEQ ID NOs: 40, 39, 36, 64 and 69, in particular SEQ ID NO: 40.

The invention also relates to a pharmaceutical composition intended (and/or suitable) for topical application to the skin, administration by inhalation or other administration to the lungs and/or oral administration, rectal administration or other route of administration to the gastric tract, containing a TNF binding agent , according to the invention, which is essentially in a monovalent format (and preferably as a D or E1D mutation at position 1 and a C-terminal extension such as C-terminal alanine). Again, said TNF binding agent is preferably selected from the group consisting of SEQ ID NOs: 40, 39, 36, 64, 69, 37, 38, 41, 62-63 and 65-68, in particular SEQ ID NOs: 40, 39, 36, 64 and 69, in particular SEQ ID NO: 40.

The invention also relates to a method for the prevention or treatment of a skin disease or disorder, which method comprises applying to the skin of a subject in need of such treatment a TNF binding agent of the invention that is in substantially monovalent format (and preferably as a D or E1D at position 1 and a C-terminal extension such as C-terminal alanine) or a composition containing such a monovalent TNF binding agent.

The invention also relates to a method for preventing or treating a disease or disorder of the skin, which method comprises administering (for example, by inhalation) to the lungs of a subject in need of such treatment, a TNF binding agent of the invention, which is essentially in a monovalent format (and preferably as a D or E1D mutation at position 1 and a C-terminal extension such as C-terminal alanine), or a composition containing such a monovalent TNF binding agent.

The invention also relates to a method for the prevention or treatment of diseases or disorders of the gastrointestinal tract, such as inflammatory bowel disease (IBD), Crohn's disease, irritable bowel syndrome, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, trauma of the digestive tract and/or cancer of the digestive tract, in in particular IBD or Crohn's disease, which method comprises administering (e.g., oral or rectal administration) to the gastrointestinal tract of a subject in need of such treatment a TNF binding agent of the invention, which is essentially in a monovalent format (and preferably as a D or E1D mutation at position 1 and a C-terminal extension such as C-terminal alanine) or a composition containing such a monovalent TNF binding agent.

As mentioned previously, the mucosal barrier of the digestive tract is often compromised at sites of inflammation, allowing orally administered proteins to enter the intestinal tissues and systemic circulation. In one embodiment, the invention also relates to a composition, compound, construct, TNF binding agent, polypeptide, or ISVD as described herein, wherein the composition, compound, construct, TNF binding agent, polypeptide, or ISVD reaches the intestinal tissues and systemic circulation in a patient .

Other aspects, embodiments, advantages and applications of the invention will become apparent from the following description.

Hereinafter, the present invention will be described using the following non-limiting preferred aspects, examples and figures, in which:

- Figure 1 is a table listing some amino acid positions that will be specifically mentioned here and their numbering according to some alternative numbering systems (such as Aho and IMGT);

- Figure 2 shows the alignment of SEQ ID NOs: 1, 59 and 58;

- Figure 3 shows the amino acid sequences specified in this application;

- Figure 4 shows the alignment of SEQ ID NOs: 1, 8-41 and 58;

- Figure 5 shows the alignment of SEQ ID NOs: 1, 31 and 36-41;

- Figure 6 shows two corresponding plots of data points obtained in Example 1 when 96 serum samples (66 from healthy people and 30 from SLE patients) were tested for binding to the following amino acid sequences: SEQ ID NO: 58 (with Q108L mutation), Reference A, Reference A (L11V, V89L)-Ala, Reference A (L11V, A74S, V89L)-Ala and Reference A (L11V, S49A, V89L)-Ala. Each dot represents the level of binding for one of the 96 tested samples. The data points shown in the right panel and left panel are the same; the right panel measures the data determined with each individual sample for each of the compounds tested (e.g. SEQ ID NO: 58 (Q108L), Reference A, Reference A (L11V, V89L)-A, Reference A (L11V, A74S , V89L) -A and Reference A (L11V, S49A, V89L)-A) are linked by a line (as a result, the slope of the line indicates the extent to which the binding of pre-existing antibodies is reduced by introducing the mutation of the invention and C-terminal alanine);

- Figure 7 is a table listing the binding data (5 columns giving normalized Pred.At binding levels (Rel.un. at 125 seconds) and 4 columns giving percentage reduction in Pred.At binding compared to Reference A from points data presented in Figure 6).

- Figure 8 (A) Shows Prognostic ("P") and Experimental ("E") trypsin and chymotrypsin cleavage sites based on the A016600015 sequence. (B) Analysis of A016600015 on a Coomassie-stained SDS-PAGE gel. (C) Exemplary result of trypsin digestion analysis of sequence A016600015 by RPQ LC-MS.

- Figure 9 is a schematic representation of the SHIME model.

- Figure 10 - Competitive FACS analysis on HEK293H-mTNF cells treated with Enbrel (EC30 = 0.02 nM, panel A) and Enbrel (EC90 = 0.2 nM, panel B). IRR000027 = Irrelevant nanobody.

- Figure 11 shows the results of expression of A016600021, A016600039 and A016600040 on a 12% NuPage Bis-Tris gel under non-reducing conditions, with 5 µl of sample applied. The lanes were as follows: (1) A016600021; (2) A016600039; (3) A016600040; (4) irrelevant comparator; (5) irrelevant comparator; (6) Precision Plus protein standard (BioRad); (7) Standard 0.5 µg; (8) Standard 1.0 μg; (9) Standard 2.5 µg.

Examples

The present inventors have set themselves the task of optimizing the amino acid sequence ("Sequence Optimization") of TNF binding agents. In this case, the TNF binding agents are intended for oral administration, so the TNF binding agents should preferably be resistant to proteases. In addition, the sequence optimization process also contemplates (1) humanizing TNF binding agents; (2) inactivate potential epitopes of preexisting antibodies; and (3) inactivate sites for post-translational modifications (PTM). At the same time, these characteristics must be brought into line with the functional characteristics of TNF binding agents, ie TNF-α inhibition, which should preferably be about the same or even improved.

Example 1 Preparation of Nanobody in Pichia pastoris and Purification by Protein A Coupling.

Pichia pastoris X33 cells containing anti-TNFα nanobody constructs were grown (30°C, 250 rpm) in 24-well plates (24 ml) in BGCM-citrate buffer. Two days later, the medium was changed to buffer containing methanol (BMCM-citrate) to induce expression. Fresh methanol was added on a regular basis to compensate for methanol consumption and evaporation, and the medium was collected two days later. The nanobodies were purified by capture on a Protein A (Poros) or MEP Hypercel (Pall) column, followed by elution in glycine buffer, according to the manufacturer's instructions. The nanobodies were then desalted in PBS using a 2 ml Zebaspin column (Pierce). Fractions were concentrated using VivaSpin columns (MWCO 5000, PES). Nanobody fraction concentrations were measured using Trinean Dropsense. Concentration was based on OD280 measurement with normalization of OD340 values. The purity and integrity of the nanobodies were verified by SDS-PAGE and MS using a reverse phase HPLC system coupled to a quadrupole electrospray ionization time-of-flight mass spectrometer (Q-TOF Ultimate (Waters).

Example 2. Humanization

The protein sequences of the TNF binding agents of the present invention were ultimately derived from llamas, and differed in part from homologous antibodies naturally occurring in humans. Therefore, these TNF binding agents are potentially immunogenic when administered to human patients.

Typically, for purposes of humanization, nanobody sequences are made more homologous to a human germline consensus sequence. With the exception of the "Hallmark" residues of the nanobodies, specific amino acids in the framework regions that differ between the consensus sequence of the nanobody and the human germline are replaced with the human counterpart in such a way that protein structure, activity and stability are preferably preserved intact.

In this case, after alignment with the human germline V gene database, DP51 was identified as having the highest homology to SEQ ID NO:58. All possible permutations were designed to change the original nanobody sequence to more closely match the human germline DP51 consensus sequence while maintaining the preferred characteristics of other nanobodies, or even improving those characteristics.

As a result, in SEQ ID NO: 58: 1E, 14P, 27F, 29F, 40A, 49S, 73N, 75K, 78L, 82aN, 83R and 108L, a total of 12 amino acid residues were introduced. Notably, Q27F and S29F are part of CDR1 but do not affect binding (see SEQ ID NO: 1, data not shown).

Example 3 Reduced Binding of Preexisting Antibodies

3.1. experimental part

The human samples used in Example 3.2 below were obtained from either commercial sources or human volunteers (after obtaining all necessary consents and approvals) and were used in accordance with applicable legal and regulatory requirements (including those relating to medical privacy and confidentiality of the patient, but not limited to).

In Example 3.2 below, unless explicitly stated otherwise, the binding of pre-existing antibodies that are present in the samples used (i.e., from healthy volunteers, rheumatoid arthritis (RA) patients, and SLE patients) to the nanobodies tested was determined using ProteOn as follows:

The nanobodies were captured with either serum albumin or FLAG3 label using the anti-FLAG M2 monoclonal antibody.

In the case of binding of preexisting antibodies to nanobodies captured on human serum albumin (HSA), evaluation was performed using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphate buffered saline, pH 7.4, 0.005% Tween20) was used as electrode buffer and experiments were performed at 25°C. ProteOn GLC sensor chip ligand bands were activated with EDC/NHS (flow rate 30 μl/min) and HSA was injected at 10 μg/ml in ProteOn acetate buffer, pH 4.5 (flow rate 100 μl/min) to provide immobilization levels approximately 3200 Rel.u. After immobilization, surfaces were decontaminated with ethanolamine-HCl (flow rate 30 μl/min). The nanobodies were injected for 2 minutes at 45 μl/min over the HSA surface to provide a capture level of the nanobodies of about 200 Rel.u. Samples containing pre-existing antibodies were centrifuged for 2 minutes at 14,000 rpm and the supernatant was diluted 1:10 in PBS-Tween20 (0.005%) before injection for 2 minutes at 45 µl/min followed by a dissociation step 400 seconds. After each cycle (ie, before the new nanobody capture step and blood sample injection step), HSA surfaces were regenerated with a 2 minute injection of HCl (100 mM) at 45 μl/min. Sensorgram processing and data analysis were performed with ProteOn Manager 3.1.0 software (Bio-Rad Laboratories, Inc.). Sensorgrams showing binding of preexisting antibodies were obtained after double comparison with the reference by subtracting (1) dissociation of the nanobody-HSA and (2) non-specific binding to the reference ligand lane. The binding levels of preexisting antibodies were determined by setting the reporting points at 125 seconds (5 seconds after the end of association). The percentage reduction in binding of preexisting antibodies was calculated relative to binding levels at 125 seconds for the reference nanobody.

In case of binding of pre-existing antibodies to FLAG-labeled nanobodies captured on monoclonal anti-FLAG M2 (Sigma), evaluation was performed using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphate buffered saline, pH 7.4, 0.005% Tween 20) was used as electrode buffer and experiments were performed at 25°C. ProteOn GLC Sensor Chip ligand bands were activated with EDC/NHS (flow rate 30 μl/min), anti-FLAG M2 mAb was injected at a concentration of 10 μg/ml in ProteOn acetate buffer, pH 4.5 (flow rate 100 μl/min) for ensuring the level of immobilization of approximately 4000 Rel.ed. After immobilization, the surfaces were deactivated with ethanolamine-HCl (flow rate 30 μl/min). The nanobodies were injected for 2 minutes at a rate of 45 μl/min over the surface of the anti-FLAG M2 to ensure a capture level of nanobodies of approximately 100 rel.u. To reduce non-specific binding of blood samples to the anti-FLAG M2 surface, 100 nM 3xFLAG peptide (Sigma) was added to blood samples. Samples containing preexisting antibodies were centrifuged for 2 minutes at 14,000 rpm and the supernatant was diluted 1:10 in PBS-Tween20 (0.005%) before administration for 2 minutes at 45 µl/min followed by a dissociation step for 600 seconds. After each cycle (i.e. before the new nanobody capture step and the blood sample injection step), anti-FLAG M2 surfaces were regenerated with a 10 second injection of glycine, pH 1.5 (10 mM) at 150 μl/min. Sensorgram processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensorgrams showing the binding of preexisting antibodies were obtained after double comparison with the reference by subtracting (1) dissociation of the anti-FLAG M2 nanobody and (2) non-specific binding to the reference ligand lane. The binding levels of preexisting antibodies were determined by setting the reporting points at 125 seconds (5 seconds after the end of association). The percentage reduction in binding of preexisting antibodies was calculated relative to binding levels at 125 seconds for the reference nanobody.

3.2. Introduction of mutations of the invention into Reference A (SEQ ID NO: 1) results in reduced binding of pre-existing antibodies.

The following amino acid sequences were used: SEQ ID NO: 58 (with mutation Q108L), Reference A, Reference A (L11V, V89L) -Ala, Reference A (L11V, A74S, V89L) -Ala and Reference A (L11V, S49A , V89L) -Ala, all with N-terminal tag HIS6-FLAG3 (SEQ ID NO: 42). These nanobodies were tested for binding to preexisting antibodies that are present in serum samples from 96 healthy human volunteers. Compounds were captured using the FLAG marker and binding was measured using ProteOn according to the protocol given in the preamble to this experimental part.

The results are shown in Figure 6. Figure 7 shows the results for each of the samples that form one of the data points in Figure 6.

It can be seen that for most of the 96 samples tested, the introduction of mutations according to the invention results in a decrease in the binding of preexisting antibodies, the extent of the decrease generally depending on the level to which the preexisting antibodies in each sample were able to bind to Reference A.

Example 4 Chemical Stability Analysis

The chemical stability of various humanized and/or optimized nanobodies was evaluated using accelerated oxidation and temperature stress assays.

A new reference compound was obtained, ie A016600015 (SEQ ID NO: 61). This new reference compound is more in line with putative clinical candidates, and thus allows a better assessment of the impact of mutations. A016600015 is identical to Reference A (SEQ ID NO: 1) except for the C-terminal alanine and aspartate at amino acid residue 1. The C-terminal alanine was introduced to reduce preexisting antibodies according to WO 2012/175741 (see Example 3). The N-terminal glutamate was replaced with aspartate at amino acid residue 1 after evaluation of pyroglutamate formation. The activity of the new reference A016600015 was almost identical to Reference A (data not shown). This new compound A016600015 is used as the reference compound in all examples unless otherwise indicated.

Based on the results of Example 3, L11V and V89L anti-PEA mutations were introduced (among others) compared to the new reference compound A016600015, resulting in A016600018 (SEQ ID NO: 37) and A016600019 (SEQ ID NO: 38) . The sequences are shown in Figure 3.

4.1. Stability under accelerated oxidation

Nanobody samples of reference compound A016600015 (1 mg/mL) were exposed for four hours at room temperature and in the dark to 10 mM H ₂ O ₂ in PBS, in parallel with controls without H ₂ O ₂ , followed by PBS buffer exchange using desalting agents. Zeba centrifuge columns (0.5 ml) (Thermo Scientific). The stressed and control samples were then analyzed by reverse phase chromatography on a 1200 or 1290 series apparatus (Agilent Technologies) on a Zorbax 300SB-C3 column (Agilent Technologies) at 70°C. The oxidation of nanobodies was quantified by analyzing the area % of previous peaks resulting from oxidative stress compared to the main protein peak.

No changes were observed in samples subjected to oxidative stress from reference compound A016600015 (data not shown).

4.2. Stability under thermal stress

Nanobody samples (1-2 mg/ml) were stored in PBS for four weeks at -20°C (negative control), 25 and 40°C. After this incubation period, the nanobodies were digested with trypsin or LysC. Stressed and control peptides were then analyzed by reverse phase chromatography on a 1290 series apparatus (Agilent Technologies) on an Acquity UPLC BEH300-C18 column (Agilent Technologies) at 60°C. The column was connected to a Q-TOF mass spectrometer (6530 Accurate Mass Q-TOF (Agilent)). Integration of a UV 214 nm peptide map or EIC chromatograms (extracted ion chromatograms) allows a reliable quantification of this modification.

Only at 40°C some isomerization and pyroglutamate variants were observed. It is noteworthy that amino acid residue 54D, which is located in CDR2, exposed to the environment and potentially amenable to isomerization, was practically not subjected to isomerization. The isomerization of amino acid residue 1 was negligible if the nanobodies were maintained at or below 25° C. for extended periods of time.

4.3. Melting Points in Thermal Shift Analysis (ATS)

The melting point of a nanobody is a measure of its biophysical stability.

The melting points of various nanobodies were evaluated by the thermal shift method (ATS), essentially according to Ericsson et al. 2006 (Anals of Biochemistry, 357: 289-298). Briefly, 5 µl of purified monovalent nanobodies (800 µg/ml) were incubated with 5 µl of Sypro Orange fluorescent probe (Invitrogen, S6551) (final concentration 10×) in 10 µl of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, 115 mM NaCl buffered at different pH from 3.5 to 9). The samples were heated in a LightCycler 480II (Roche) from 37 to 99°C at a rate of 4.4°C/s, after which they were cooled to 37°C at a rate of 0.03°C/s. Heat-induced unfolding exposes the hydrophobic portions of the proteins to which Sypro Orange binds, resulting in an increase in fluorescence intensity (Ex/Ev = 465/580 nm). The inflection point of the first derivative of the fluorescence intensity curve serves as a measure of the melting temperature (Tm).

Reference compound A016600015 (SEQ ID NO: 61) was compared with A016600018 (SEQ ID NO:37) and A016600019 (SEQ ID NO:38). In contrast to A016600015, both A016600018 and A016600019 contain anti-PEA mutations L11V and V89L, in accordance with Example 3.

The results are shown in Table 4.3.

Table 4.3

NB ID Anti-PEA Quantity (mcg) Tp (°C), pH 7 A016600015 - 87 59 A016600018 L11V V89L 38 59 A016600019 L11V V89L 16 56

As can be seen from Table 4.3, anti-PEA mutations seem to either not affect Tp (A016600018) or negatively affect Tp (A016600019). Notably, the difference between A016600018("00018") and A016600019("00019") is 78L and 78V, respectively. In general, any effects of this difference in amino acid residue 78 were shown to be negligible compared to the introduction of anti-PEA mutations (see also below).

During manufacture, it was also observed that the expression of these variants containing anti-PEA mutations is suboptimal. This was further quantified using the method of Example 1.

The results are shown in Table 4.3.

Indeed, the amount recovered from both variants containing anti-PEA mutations was about 2 times lower (A016600018) or even 4 times lower (A016600019) than that of the reference nanobody. This came as a complete surprise to the inventors, since the earlier introduction of these L11V and V89L anti-PEA mutations did not lead to such a severe reduction in the ability to extract antibodies, which was determined for any other clone.

4.4. Protease resistance

It is contemplated that TNF inhibitors may eventually be administered orally. However, the stomach and intestines are a natural hostile environment as they are designed to enzymatically break down and absorb partially solid foods. In general, proteolysis of a protein substrate can only occur if 8-10 amino acid residues within the polypeptide chain can bind and adapt to the specific stereochemistry of the active site of the protease (Fontana et al., 2004 Acta Biochim Pol, 51, 299-321). Therefore, susceptibility to enzymatic degradation depends largely on the physical properties of the substrate.

In order to identify potential protease degradation sites and develop more stable variants, the present inventors set out to identify trypsin and chymotrypsin cleavage sites according to standard methods.

The predicted protease sites of action ("P") of SEQ ID NO: 58 and the new reference compound A016600015 ("00015") are depicted as ("X") in Figure 8A for both trypsin and chymotrypsin.

From this figure, we can already conclude that:

- CDR3 contains 9 potential protease cleavage sites.

the humanizing mutation Q75K (VH3-DP51) introduces a potential trypsin cleavage site.

- humanizing mutation K73N (VH3-DP51) eliminates a potential trypsin cleavage site.

- anti-PEA mutations L11V and V89L were neutral in this respect, ie these mutations did not introduce or eliminate potential protease cleavage sites, as expected.

To assess the predicted sites of protease cleavage under conditions close to in vivo, nanobodies were cleaved with trypsin and chymotrypsin. In particular, anti-TNFα nanobodies were incubated in 10% trypsin or α-chymotrypsin solutions. Two μg of the nanobodies were subjected to trypsin digestion for 2 h, 4 h and overnight at 37°C, or chymotrypsin digestion for 2 h, 4 h or overnight at 25°C. The proteolytic reaction was stopped by adding TFA (to a final concentration of 0.1%). Reaction mixtures were analyzed by reverse phase liquid chromatography-mass spectrometry or separated on gel in SDS-PAGE and stained with Coomassie blue. Using ImageQuant (GE), the amount of intact material was calculated and normalized to 0 hours as a time reference.

An exemplary result of trypsin digestion on a Coomassie-stained gel from SDS-PAGE is shown in Figure 8B. An exemplary result of reversed-phase LC-MS trypsin digestion analysis is shown in Figure 8C. Experimentally ("E") confirmed cleavage sites are depicted in Figure 8A as trypsin ("E") and chymotrypsin ("E").

From Figure 8A it can be seen that the number of actual cleavage sites is reduced. However, there remain distinct sites detected by trypsin and chymotrypsin.

To optimize trypsin resistance, 5 positions were selected to develop one optimized variant: R38, K64, S94, P95 and R96, resulting in variant A016600013; SEQ ID NO: 65.

However, mutation of these sites resulted in reduced levels of expression and intracellular accumulation of the variant (A016600013; SEQ ID NO: 65). In fact, when amino acid residue 38 was mutated to the original species, which improved expression, the resulting variant (A016600014; SEQ ID NO: 66) completely contradicted the expectation of greater protease sensitivity than the control compound A016600015 (data not shown). Therefore, it was decided not to mutate these positions in the control compound and nanobodies under study, ie A016600018 and A016600019.

A summary of the results is shown in Table 4.4.

Table 4.4 Intact antibody content (%) after incubation (hours) with proteases trypsin Chymotrypsin nanobody L11 V89 0 h 2 h 4 h Night 0 h 2 h 4 h Night A016600015 . . 100 95 99 79 100 16 eighteen fifteen A016600019 V L 100 83 91 78 100 27 39 31 A016600018 V L 100 89 85 63 100 38 31 fourteen

Example 5 Stability in Intestinal Juices in the SHIME Model

To investigate the persistence of anti-TNF-α nanobodies in the human GI tract, the nanobodies were incubated in 5 different fluids derived from SHIME (human gut microbiological ecosystem simulator) representing the human gastrointestinal (GI) tract. The SHIME model is a science-based dynamic model of the complete gastrointestinal tract that is used to study physicochemical, enzymatic, and microbial parameters in the gastrointestinal tract in an in vitro controlled environment.

The SHIME model consists of five reactors that sequentially simulate the stomach (acidic conditions and pepsin digestion), small intestine (digestive processes) and 3 areas of the large intestine, i.e. ascending ("A"), transverse ("T") and descending colon ("D") (microbial processes). Careful control of environmental parameters in these reactors has provided complex and stable microbial communities that are very similar in structure and function to the microbial community in different areas of the human colon (see Figure 9). All GI fluids were provided by ProDigest (Technologiepark 4, 9052 Zwijnarde, Belgium). Anti-TNF nanobodies were tested for a maximum period of 39 hours in gastrointestinal fluids. After incubation in gastrointestinal fluids, the stability of the nanobodies was determined by functionality testing in a competitive ELISA. The nanobodies were tested at a fixed concentration of 100 μg/ml at 37° C. in 5 different GI fluids in the SHIME model. Samples were taken at different time points and stored at -20°C with or without the addition of protease inhibitors, according to the schedule in Table 5.1. Samples were transferred to ELISA coated plates and subsequently tested in ELISA. Briefly, A016600015 was applied at a concentration of 1 μg/ml in PBS. After blocking, a fixed concentration of 0.3 nM biotinylated hTNFα was added along with a series of titrations of variants in various GI fluids. Detection was carried out with extravidin - horseradish peroxidase

Table 5.1. Incubation calendar.

Liquid Moments in time TC on an empty stomach 0, 1, 2, 4, 6, 8 hours* TC after
food intake 0, 1, 2, 4, 6, 8 hours* Gut A 0, 1, 2, 4, 8, 15, 20, 24 hours Gut T 0, 1, 2, 4, 8, 15, 20, 24, 39 h Gut D 0, 1, 2, 4, 8, 15, 20, 24, 32 hours PBS 0, 1, 2, 4, 6, 8, 15, 20, 24, 32 hours

*after incubation with the addition of 1 mg/ml pefabloc and 1 μM pepstatin (protease inhibitors).

Exemplary results in the "T" colon are shown in Table 5.2.

Table 5.2

0 h 1 hour 2 h 4 h 8 h 15 h 20 h 24 hours 39 h 00015 _IC50 7.3E-10 5.0E-10 6.4E-10 6.2E-10 9.7E-10 1.3E-09 1.9E-09 2.8E-09 2.0E-08 Relation 1.00 0.69 0.88 0.85 1.34 1.72 2.56 3.88 27.41 % 100% 145% 113% 117% 75% 58% 39% 26% four% _IC50 5.7E-10 5.5E-10 6.9E-10 1.2E-09 2.8E-09 9.5E-09 3.2E-08 4.2E-08 6.2E-07 00018 Relation 1.00 0.97 1.22 2.19 4.96 16.68 55.58 73.79 1097.18 % 100% 103% 82% 46% twenty% 6% 2% one% 0% _IC50 5.0E-10 4.7E-10 4.5E-10 9.1E-10 7.2E-10 1.7E-09 3.0E-09 5.2E-09 4.1E-08 00019 Relation 1.00 0.93 0.89 1.81 1.43 3.36 6.02 10.32 82.22 % 100% 108% 112% 55% 70% thirty% 17% ten% one%

Based on results with fluids obtained from different SHIME departments, the stability score allows nanobodies to be ranked, representing stability in all GI regions: 00015 > 00019 > 00018.

Taken together, the elimination of protease cleavage sites does not appear to have a beneficial effect on, for example, protease stability and sensitivity for this family of nanobodies. The introduction of anti-PEA mutations L11V and V89L in A16600015 appears to adversely affect the stability of this particular nanobody.

Example 6 Stabilized Variants

Since the removal of cleavage sites by proteases did not lead to the expected results, the inventors further developed complex variants of anti-TNF-α, which would be inherently more stable, but in which humanizing and anti-PEA mutations are preferably not disturbed. This required an unconventional approach considering different amino acid residues showing mutually exclusive characteristics such as humanization versus protease resistance versus affinity.

6.1. Internal cysteine bonding and elimination of the CDR3 chymotrypsin cleavage site.

Although Y100d is located in CDR3 and potentially affects binding properties, it was nevertheless decided to construct a variant in which the Y100d amino acid residue, which is the chymotrypsin cleavage site, was removed by replacing Y100dL (A016600046 100dL; SEQ ID NO: 62). Variant A016600045 (SEQ ID NO: 69) was designed to evaluate the effect of tyrosine on position 100d of CDR3.

In addition, the inventors have suggested that variants that are inherently stable can be constructed by introducing an intradomain disulfide bond (see Wozniak-Knopp et al., 2012 PLoS One. 2012; 7 (1): e30083) . This would require the introduction of two cysteine residues, which would then form a cystine. After resolving the protein structure of the nanobody and its interaction with the TNF-α target using crystallization studies (data not shown), the inventors decided to mutate the amino acids S49C and I69C mutations to introduce an intradomain disulfide bond, although the S49 amino acid residue is located adjacent to CDR2, and was introduced for the purpose of humanization (corresponding to human germline DP51, see Example 2). The new variant containing S49C, I69C and Y100dL is designated as A016600052 (SEQ ID NO: 63).

To fully evaluate the effect of the position of amino acid 49, this amino acid residue was replaced with alanine. Notably, this mutation at position 49 would correspond (albeit different) to the human IgHV3-IgHJ germline. All new variants A016600046 (SEQ ID NO: 62), A016600016 (SEQ ID NO: 36), A016600020 (SEQ ID NO: 39) and A016600021 (SEQ ID NO: 40) contain A49.

In addition, in version A016600020 (SEQ ID NO: 39), positions D60A, E61D and P62S were evaluated in terms of physical stability. Amino acid residues A60, D61 and S62 are located near the experimentally confirmed Y58 chymotrypsin cleavage site and within CDR2 according to Kabat, implying a high risk of possible loss.

Further, a protease cleavage site was accidentally introduced by the Q75K humanizing mutation. Without wishing to be bound by any theory, the inventors hypothesized that altering the adjacent amino acid residue A74S would potentially affect both humanization and protease sensitivity, but only to a certain extent, i.e. they hoped that both humanization and protease sensitivity decreased to an acceptable degree. Variants including S74 are A016600016 ("00016"), A016600020 ("00020"), A016600021 ("00021"), A016600046 ("00046") and A016600052 ("00052") (SEQ ID NO 36, 39, 40 , 62 and 63, respectively). The comparative variant was A016600038 ("00038", SEQ ID NO: 64), which is identical to A016600021 (SEQ ID NO: 40) but with 74S instead of 74A.

The resulting sequences are shown in Figure 3.

6.2. Characteristics of stabilized variants

These variants were then evaluated for various characteristics essentially as indicated in Examples 3 and 4.

In the first case, the production of variants 00016, 00020 and 00021 was determined in P. pastoris in accordance with Example 1.

A summary of the results obtained is given in Table 6.2A.

Table 6.2A

nanobody L11 S49 D60 E61 P62 A74 L78 V89 Quantity (mcg) Tp at pH 7 (°C) Stability in gastrointestinal fluid (ascending) A016600021 V A . . . S V L 332 65 one A016600016 V A . . . S . L 499 65 2 A016600020 V A A D S S V L 374 75 3

From these results, it can be seen that these mutations resulted in an increase in production of more than 4-5 times, compared with the preparation of the reference compound, which was obtained in an amount of 87 μg (see Example 4.3) or even more than 20-30 times, compared to the closely related variants 00018 and 00019.

Then the thermal stability of these options was determined in accordance with Example 4.3. A summary of the results obtained is also presented in Table 6.2A.

Not only did the production increase dramatically, but the thermal stability unexpectedly increased from 5-16°C compared to the reference compound, and even up to 16-19°C compared to the closer variants 00018 and 00019.

Surprisingly, amino acid residue A49 abolished the effect of anti-PEA amino acid residues V11 and L89 on production and stability.

The different variants from Example 6.1 were evaluated together in GI fluids obtained on the SHIME model according to Example 5.

A summary of the activity obtained at the end of the incubation period under various conditions A016600021, A016600038, A016600046 and A016600052 is shown in Table 6.2B. Incubation in PBS confirmed the inherent stability of the nanobodies (data not shown).

Table 6.2B
C/C0 against time, in hours BK gut A BK gut D BK gut T TC on an empty stomach * TC after meals* YAK2 gut A YAK2 gut D YAK2 gut T A016600021 66 58 76 65 31 86 57 71 A016600038 48 41 47 60 33 77 fifty 59 A016600046 47 29 62 52 24 92 59 >100 A016600052 41 49 40 ND ND 64 43 55 A016600039 38 26 39 23 twenty 78 twenty 55 A016600040 38 40 19 67 24 91 83 63 A016600045 85 >100 49 60 26 68 41 >100

* with pathology; ND - not determined; CD - Crohn's disease; UC - ulcerative colitis.

Variants A016600046 Y100dL (SEQ ID NO: 62), in which the protease cleavage site was removed, and variant A016600052 (SEQ ID NO: 63), which was further stabilized with an intradomain cysteine bond, did not provide any improvement in this regard. Notably, the IC50 of variants 00046 and 00052 increased 2-5 times compared to 00021 (1.13 nM and 3.56 nM, respectively, compared to 0.67 nM) due to a mutation in CDR3.

The results of SHIME substantially confirmed and extended the results of production and Tp. In addition, the SHIME model showed that variant 00021 (SEQ ID NO: 40) is significantly more stable than variant A016600040 ("00040", SEQ ID NO: 67) and variant 00038 (SEQ ID NO: 64), which is indicative of positive contribution of serine at position 74.

Notably, variant A016600039 (SEQ ID NO: 68), which is identical to variant 00040 (SEQ ID NO: 67) but with A74S, showed that S74 was significantly less stable than A74 on the SHIME model.

Based on all results, including results with fluids obtained from different departments of SHIME, the overall stability score allows nanobodies to be ranked, representing stability in all departments of the gastrointestinal tract: 00021 > 00016 > 00020 > 00040 > 00015 > 00019 > 00018.

Therefore, the L11V and V89L anti-PEA mutations in A16600015 appear to have a negative effect on the stability of this particular nanobody, which can be reduced by 49A but not by 49C-69C. Moreover, in nanobodies containing mutations against PEA, L11V and V89L, the 74S amino acid residue was favorable for stability in the SHIME model.

6.3. A016600021 has favorable characteristics (compared to A016600039 and A016600040)

It is believed that for oral administration, in addition to stabilization, a higher dose may be required. In order to have acceptable costs, the candidate is preferably obtained in large quantities, purified without significant loss, and prepared at a high concentration. Therefore, the inventors carried out experiments to test these parameters.

First, the inventors decided to compare the expression levels of A016600039 and A016600040 with A016600021.

As can be seen in Figure 11A, the expression of A016600039 (SEQ ID NO: 68) was significantly lower than the expression levels of A01660021 (SEQ ID NO: 40) and A016600040 (SEQ ID NO: 67). These data were also quantitatively analyzed using AKTAmicro, the results of which are shown in Table 6.3.

Table 6.3

Clone Yield of Aktaµ (g/l cell-free medium) A016600021 6.3 A016600039 0.49 A016600040 2.3

With regard to the workability of A016600021 compared to A016600040, both were tested for maximum solubility during concentration of the fermentation broth. The maximum solubility in the purified fermentation broth is only 30 g/l for A016600040 (versus 75 g/l for A016600021). In addition, significant losses were found during further purification of A016600040.

Therefore, in addition to stability, A016600021 also has unexpectedly favorable production and cleaning characteristics compared to A016600039 and A016600040.

Based on the advantageous characteristics of stability, expression and purification, the inventors focused on A016600021, for which the solubility was determined. To the complete surprise of the inventors, A016600021 had a solubility up to an unprecedented amount of 200 mg/ml in H ₂ O.

Example 7 Stabilized Variants Outperform Benchmarks

Variants A016600021 (SEQ ID NO: 40), A016600038 (SEQ ID NO: 64) and A016600045 (SEQ ID NO: 69) were evaluated for binding to human membrane-bound TNF (mTNF), in a competitive assay with Enbrel (Etanercept), anti-TNF therapeutic agent based on TNF-R2. To this end, a cell line stably expressing a non-cleavable form of human TNF was obtained by transfecting HEK293H cells with a eukaryotic expression vector encoding a human TNF R77T/S78T variant (referred to as HEK293H-mTNF) as previously described (Harashima et al., 2001 J. Immunol 166: 130-136). After selection by co-expression of the puromycin resistance gene, sorting of individual mTNF-expressing cells was performed via FACS (BD FACS Aria) by staining cells with Remicade (Infliximab) and secondary goat F(ab') ₂ antibodies against human IgG, mouse antibodies labeled FE. Constitutive expression of membrane-bound TNF in selected individual clones was confirmed by flow cytometry (BD FACS Array).

In the first case, the binding of Enbrel to mTNF in these cells was evaluated. For this, 5.10 ⁵ cells of one HEK293H-mTNF clone were seeded in 96-well round bottom plates and directly incubated with different concentrations of Enbrel diluted in FACS buffer (PBS supplemented with 10% FTS and 0.05% sodium azide) for 90 minutes at 4°C. Cells were washed with FACS buffer and stained with a secondary goat F(ab') ₂ antibody against human IgG, a PE-labeled mouse antibody, for 30 minutes at 4°C. After washing and incubating cells in FACS buffer in the presence of dead cell dye TO-PRO-3 Iodide, Enbrel binding to mTNF in viable HEK293H-mTNF cells was measured by reading on the BD FACS Array. From the resulting dose-response curve, the EC30 and EC90 values for Enbrel for mTNF binding were determined to be 0.02 nM and 0.2 nM, respectively. Both concentrations were subsequently used in a competitive assay with TNF-targeted nanobodies.

To assess the binding of mTNF A016600021, A016600038 and A016600045, a competition experiment was performed with Enbrel at fixed concentrations of EC30 and EC90. The anti-TNF activity of the nanobodies was compared with the control compound Cimzia (certolizumab pegol). In addition, an irrelevant nanobody (IRR00027) was included as a negative control. In this assay, identical conditions were used as described above for Enbrel binding, except for the first incubation step, which in this experiment involved co-treating the cells with a fixed concentration of Enbrel (EC30 or EC90) in combination with various concentrations of anti-TNF nanobodies or Cimzia from 300 nm up to 12.5 pM. The BD FACS array readings determined Enbrel binding on HEK293H-mTNF cells, which was clearly reduced by co-treatment with increasing nanobodies concentration, as well as with Cimzia, indicating competition of anti-TNF agents with Enbrel for binding to mTNF. IC50 values of anti-TNF agents were determined as indicated in Table 7.

Table 7 Enbrel (EC30: 0.02 nM) Enbrel (EC90: 0.2 nM) A016600021 0.43 nM 0.65 nM A016600038 0.43 nM 0.70 nM A016600045 0.54 nM 0.76 nM Cimzia 7.9 nM 4.8 nM

The results obtained show that A016600021, A016600038 and A016600045 are 6-7 times more efficient than Cimzia on EC90 and a surprising 15-18 times better than Cimzia on EC30. The stabilized variants have comparable potency to compete with Enbrel for mTNF binding.

In competitive FACS, it was demonstrated that nanobodies can also completely inhibit Enbrel binding to mTNF. This may be indicative of superior suitability as an oral drug. In contrast, high concentrations of Cimzia resulted in only a partial blockage of Enbrel binding (Figure 10).

The entire content of all references (including references to the literature, published patents, published patent applications and simultaneously pending patent applications) cited throughout this application are expressly incorporated by reference, in particular for the teachings mentioned above.

Table A: Reference A and its CDRs and PMP6C11

SEQ
ID
No. Description Subsequence 58 NC55TNF-NC7 (PMP6C11). WO06/122786: SEQ ID NO: 125 QVQLVESGGGLVQAGGSLRLSCTASGQTSSTADMGWFRQPPGKGREFVARISGIDGTTYYDEPVKGRFTISRDKAQNTVYLQMDSLKPEDTAVYYCRSPRYADQWSAYDYWGQGTQVTVSS 59 WO2015/173325 SEQ ID NO: 345 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVSRISGIDGTTYYDEPVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS one Reference A: EVQLVESGGGLVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVSRISGIDGTTYYDEPVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCRSPRYADQWSAYDYWGQGTLVTVSS 2 CDR1 (Kabat) TADMG 3 CDR2 (Kabat) RISGIDGTTYYDEPVKG four CDR3 (Kabat/Abm) PRYADQWSAYDY 5 CDR1 (Abm) GFTFSTADMG 6 CDR2 (Abm) RISGIDGTTY 7 CDR3 (Kabat/Abm) PRYADQWSAYDY Note: SEQ ID NO: 4 is identical to SEQ ID NO: 7

Table B. Possible combinations of amino acids at positions 11, 89, 110, and 112.

Table B-1: Possible combinations of amino acids at positions 11, 89, 110, 112, 49 and 74.

Table B-2: Possible combinations of amino acids at positions 11, 89, 110, 112, 49 and 74.

Table C-1: Schematic presentation of some compounds of the invention without ISVD increasing half-life.

[TNF binding agent of the invention] [TNF binding agent of the invention]-X(n) [TNF Binding Agent of the Invention]-[TNF Binding Agent of the Invention] [TNF binding agent of the invention]-[TNF binding agent of the invention]-X(n) [TNF binding agent of the invention]-[TNF2] [TNF binding agent of the invention]-[TNF2]-X(n) [TNF2]-[TNF Binding Agent of the Invention] [TNF2]-[TNF binding agent of the invention]-X(n) [TNF binding agent of the invention]-[Targeting unit] [Targeting Unit]-[TNF Binding Agent of the Invention]- [TNF binding agent of the invention]-[Targeting unit]-X(n) [TNF binding agent of the invention]-[TNF binding agent of the invention]-[Targeting unit]-X(n) Designations:
- "[TNF binding agent of the invention]" represents a TNF binding agent of the invention.
- "-" represents either a direct covalent bond or a suitable linker such as a 9GS, 15GS or 35GS linker.
- "X(n)" represents a C-terminal extension, as defined in this application, such as a single alanine residue.
- "[TNF2]" represents a binding domain or binding unit (and in particular ISVD) against TNF other than the TNF binding agent of the invention.
- "[Targeting Unit]" represents a binding domain or binding unit (and in particular, ISVD) that targets a compound of the invention to a specific cell, tissue, or organ.

Table C-2. Schematic presentation of some compounds of the invention with ISVD increasing half-life.

[TNF binding agent of the invention]-[HLE] [HLE]-[TNF Binding Agent of the Invention] [TNF binding agent of the invention]-[HLE]-X(n) [HLE]-[TNF binding agent of the invention]-X(n) [TNF Binding Agent of the Invention]-[TNF Binding Agent of the Invention]-[HLE] [TNF Binding Agent of the Invention]-[HLE]-[TNF Binding Agent of the Invention] [HLE]-[TNF Binding Agent of the Invention]-[TNF Binding Agent of the Invention] [TNF Binding Agent of the Invention]-[TNF Binding Agent of the Invention]-[HLE]-X(n) [TNF Binding Agent of the Invention]-[HLE]-[TNF Binding Agent of the Invention]-X(n) [HLE]-[TNF Binding Agent of the Invention]-[TNF Binding Agent of the Invention]-X(n) [TNF binding agent of the invention]-[TNF2]-[HLE] [TNF binding agent of the invention]-[HLE]-[TNF2] [HLE]-[TNF binding agent of the invention]-[TNF2] [HLE]-[TNF2]-[TNF Binding Agent of the Invention] [TNF2]-[TNF binding agent of the invention]-[HLE] [TNF2 -[HLE] -[TNF Binding Agent of the Invention] [TNF binding agent of the invention]-[TNF2]-[HLE]-X(n) [TNF binding agent of the invention]-[HLE]-[TNF2]-X(n) [HLE]-[TNF binding agent of the invention]-[TNF2]-X(n) [HLE]-[TNF2]-[TNF binding agent of the invention]-X(n) [TNF2]-[TNF binding agent of the invention]-[HLE]-X(n) [TNF2 -[HLE] -[TNF binding agent of the invention]-X(n) [TNF binding agent of the invention]-[Targeting unit]-[HLE] [Targeting unit]-[TNF binding agent of the invention]-[HLE] [TNF binding agent of the invention]-[Targeting unit]-[HLE]-X(n) [HLE]-[Targeting unit]-[TNF binding agent of the invention]-[HLE]-X(n) Designations:
- "[TNF binding agent of the invention]" represents a TNF binding agent of the invention.
- "-" represents either a direct covalent bond or a suitable linker such as a 9GS, 15GS or 35GS linker.
- "X(n)" represents a C-terminal extension, as defined in this application, such as a single alanine residue.
- "[HLE]" represents a domain or binding unit that increases half-life (and in particular, ISVD, which increases half-life), such as ISVD (and in particular, nanobodies) against (human) serum albumin;
- "[TNF2]" represents a binding domain or binding unit (and in particular ISVD) against TNF other than the TNF binding agent of the invention.
- "[Targeting Unit]" represents a binding domain or binding unit (and in particular, ISVD) that targets a compound of the invention to a specific cell, tissue, or organ.

Table D: Serum albumin-binding ISVD sequences ("ID" stands for SEQ ID NO as used herein).

Name ID Amino acid sequence Alb8 70 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 71 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 72 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 73 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 74 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11(S112K)-A 75 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 76 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 77 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 78 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 79 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 80 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSG Alb82-GG 81 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 82 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb92 83 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 84 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

Table E. Various Amino Acid Sequences ("ID" stands for SEQ ID NO as used herein)

Name ID Amino acid sequence 5GS linker 85 GGGGS 7GS linker 86 sggsggs 8GS linker 87 ggggcgggs 9GS linker 88 GGGGSGGGS 10GS linker 89 GGGGSGGGGS 15GS linker 90 GGGGSGGGGSGGGGS 18GS linker 91 GGGGSGGGGSGGGGGGGS 20GS linker 92 GGGGSGGGGSGGGGSGGGGS 25GS linker 93 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS linker 94 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS linker 95 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS linker 96 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 97 EPKSCDKTHTCPPCP 9GS-G1 hinge 98 GGGGSGGGSEPKSCDKTHTCPPCP Lama's upper long hinged area 99 epktpkpqpaaa G3 hinge 100 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

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SEQUENCE LIST

<110> ABLINX NV

<120> ADVANCED TNF BINDING AGENTS

<130> 192557

<150> US62/254375

<151> 2015-11-12

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 11

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 11

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Lys Ser

115 120

<210> 12

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 12

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Gln Ser

115 120

<210> 13

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 13

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Lys Val Ser Ser

115 120

<210> 14

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 14

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 15

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 15

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Lys Ser

115 120

<210> 16

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 16

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Gln Ser

115 120

<210> 17

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 17

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 18

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Lys Val Ser Ser

115 120

<210> 19

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 19

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser

115 120

<210> 20

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 20

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Lys Ser

115 120

<210> 21

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 21

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Gln Ser

115 120

<210> 22

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 22

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 23

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 23

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Lys Val Ser Ser Ala

115 120

<210> 24

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 24

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser Ala

115 120

<210> 25

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 25

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Lys Ser Ala

115 120

<210> 26

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 26

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Gln Ser Ala

115 120

<210> 27

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 27

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Lys Val Ser Ser Ala

115 120

<210> 28

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 28

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser Ala

115 120

<210> 29

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 29

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Lys Ser Ala

115 120

<210> 30

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 30

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Gln Ser Ala

115 120

<210> 31

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 31

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 32

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 32

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Lys Val Ser Ser Ala

115 120

<210> 33

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 33

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Gln Val Ser Ser Ala

115 120

<210> 34

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 34

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Lys Ser Ala

115 120

<210> 35

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 35

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Gln Ser Ala

115 120

<210> 36

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 36

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 37

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 37

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 38

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 38

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 39

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 39

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 40

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 40

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 41

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 41

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 42

<211> 34

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> HIS6-FLAG3 LABEL

<400> 42

His His His His His His His Gly Ala Ala Asp Tyr Lys Asp His Asp Gly

1 5 10 15

Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly

20 25 30

Ala Ala

<210> 43

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 43

Val Thr Val Lys Ser

fifteen

<210> 44

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 44

Val Thr Val Gln Ser

fifteen

<210> 45

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 45

Val Lys Val Ser Ser

fifteen

<210> 46

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 46

Val Gln Val Ser Ser

fifteen

<210> 47

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<220>

<221> site

<222>(6)..(6)

<223> Xaa means (X)n, which refers to the C-terminal extension

into n amino acids, where each position is independently selected from any

amino acids

<400> 47

Val Thr Val Lys Ser Xaa

fifteen

<210> 48

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<220>

<221> site

<222> Xaa means (X)n, which refers to the C-terminal extension

into n amino acids, where each position is independently chosen

from any amino acid

<400> 48

Val Thr Val Gln Ser Xaa

fifteen

<210> 49

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<220>

<221> SITE

<222>(6)..(6)

<223> Xaa means (X)n, which refers to the C-terminal extension

into n amino acids, where each position is independently chosen

from any amino acid

<400> 49

Val Lys Val Ser Ser Xaa

fifteen

<210> 50

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<220>

<221> SITE

<222>(6)..(6)

<223> Xaa means (X)n, which refers to the C-terminal extension

into n amino acids, where each position is independently chosen

from any amino acid

<400> 50

Val Gln Val Ser Ser Xaa

fifteen

<210> 51

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 51

Val Thr Val Lys Ser Ala

fifteen

<210> 52

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 52

Val Thr Val Gln Ser Ala

fifteen

<210> 53

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 53

Val Lys Val Ser Ser Ala

fifteen

<210> 54

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 54

Val Gln Val Ser Ser Ala

fifteen

<210> 55

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 55

Val Thr Val Ser Ser

fifteen

<210> 56

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<220>

<221> SITE

<222>(6)..(6)

<223> Xaa means (X)n, which refers to the C-terminal extension

into n amino acids, where each position is independently chosen

from any amino acid

<400> 56

Val Thr Val Ser Ser Xaa

fifteen

<210> 57

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> C-END

<400> 57

Val Thr Val Ser Ser Ala

fifteen

<210> 58

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 58

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 59

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 59

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 60

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 60

Asp 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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 61

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 61

Asp 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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 62

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 62

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 63

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 63

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Cys Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 64

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 64

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 65

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 65

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Pro Ser Gln Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 66

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 66

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 Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

Ser Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

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

65 70 75 80

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

85 90 95

Arg Pro Ser Gln Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 67

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 67

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 68

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 68

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 69

<211> 122

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 69

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala His Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 70

<211> 115

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 70

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 71

<211> 115

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 71

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 Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 72

<211> 116

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 72

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 73

<211> 116

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 73

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 74

<211> 115

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 74

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 75

<211> 116

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 75

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys

100 105 110

Val Ser Ser Ala

115

<210> 76

<211> 115

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 76

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 77

<211> 116

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 77

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 78

<211> 117

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 78

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ala

115

<210> 79

<211> 118

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 79

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ala Ala

115

<210> 80

<211> 116

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 80

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly

115

<210> 81

<211> 117

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 81

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly

115

<210> 82

<211> 118

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 82

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly

115

<210> 83

<211> 115

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 83

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 84

<211> 116

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 84

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 85

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 85

Gly Gly Gly Gly Ser

fifteen

<210> 86

<211> 7

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 86

Ser Gly Gly Ser Gly Gly Ser

fifteen

<210> 87

<211> 9

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 87

Gly Gly Gly Gly Cys Gly Gly Gly Ser

fifteen

<210> 88

<211> 9

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 88

Gly Gly Gly Gly Ser Gly Gly Gly Ser

fifteen

<210> 89

<211> 10

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 89

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 90

<211> 15

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 90

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 91

<211> 18

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 91

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly

1 5 10 15

Gly Ser

<210> 92

<211> 20

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 92

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 93

<211> 25

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 93

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 94

<211> 30

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 94

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 95

<211> 35

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 95

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 96

<211> 40

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Linker

<400> 96

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 97

<211> 15

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> G1 hinge

<400> 97

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 98

<211> 24

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> 9GS-G1 hinge

<400> 98

Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro

twenty

<210> 99

<211> 12

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Llama upper long hinge area

<400> 99

Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala

1 5 10

<210> 100

<211> 62

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> G3 hinge

<400> 100

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

35 40 45

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

<210> 101

<211> 5

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 101

Ser Phe Gly Met Ser

fifteen

<210> 102

<211> 17

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 102

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

1 5 10 15

gly

<210> 103

<211> 6

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> CDR SEQUENCE

<400> 103

Gly Gly Ser Leu Ser Arg

fifteen

<210> 104

<211> 121

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> TNF006C11

<400> 104

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Arg Glu Phe Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 105

<211> 4

<212> PROTEIN

<213> ARTIFICIAL SEQUENCE

<220>

<223> Label

<400> 105

Gly Gly Gly Cys

one

<---

Claims (39)

1. An immunoglobulin single variable domain (ISVD) that specifically binds TNFα, having: - CDR1 according to Abm, which is the amino acid sequence of GFTFSTADMG (SEQ ID NO: 5); and - CDR2 according to Abm, which is the amino acid sequence RISGIDGTTY (SEQ ID NO: 6); and - CDR3 according to Abm, which is the amino acid sequence PRYADQWSAYDY (SEQ ID NO: 4); - where the amino acid residue at position 49 is alanine; and where the amino acid residue at position 74 is serine - where the amino acid residues at positions 11, 89, 110 and 112 are as follows: - the amino acid residue at position 11 is selected from L or V; and - the amino acid residue at position 89 is selected from T, V or L; and - the amino acid residue at position 110 is selected from T, K or Q; and - the amino acid residue at position 112 is selected from S, K or Q; So that (i) at position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L and position 11 is V and position 110 is K or Q; or (vi) position 89 is L and position 11 is V and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (viii) position 11 is V and position 112 is K or Q; and having: - a degree of sequence identity with the amino acid sequence of SEQ ID NO: 1 of at least 90%, more preferably at least 95%, but in which a C-terminal extension that may be present, as well as CDRs and amino acid residues at positions 11, 89, 110 or 112 are not taken into account in determining the degree of sequence identity; and/or - no more than 7, for example no more than 5, preferably no more than 3, for example only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO: 1, but where the C-terminal extension that may be present, as well as the CDR and amino acid residues at positions 11, 89, 110 or 112 are not taken into account when determining the degree of sequence identity, and, if necessary, having: - C-terminal extension (X) _n in which n is 1-10, preferably 1-5, such as 1, 2, 3, 4 or 5, and preferably 1 or 2, such as 1; and each X is an amino acid residue which is preferably naturally occurring and which is independently selected and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I). 2. An ISVD according to claim 1, wherein the amino acid residue at position 73 is N and the amino acid residue at position 75 is K. 3. An ISVD according to any one of claims 1, 2, selected from the group consisting of SEQ ID NOs: 40, 39, 36, 62 and 69, in particular SEQ ID NOs: 40, 39 and 36, more specifically SEQ ID NOs: 40. 4. ISVD according to claim 3, having D in position 1. 5. ISVD according to claim 3 or 4, having a C-terminal extension X(n), which is preferably a C-terminal alanine residue. 6. A compound that specifically binds TNFα, comprising at least one ISVD according to any one of claims 1-5. 7. A compound according to claim 6, comprising at least two ISVDs according to any one of claims 1 to 5, wherein said at least two ISVDs may be the same or different. 8. The compound of claim 7, wherein said at least two ISVDs are independently selected from the group consisting of SEQ ID NOs: 8-41, 61-66 and 69, preferably 40, 39, 36, 62 and 69. 9. A compound according to any one of claims 6 to 8, comprising a serum protein binding component, wherein said serum protein binding component binds serum albumin. 10. The compound of claim 9, wherein said serum protein binding component is a serum albumin binding ISVD, and wherein said serum albumin binding ISVD essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 hypervariable regions (CDR1-CDR3, respectively), where CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. 11. The compound of claim 10 wherein said serum albumin binding ISVD is any one of SEQ ID NOs: 70-84. 12. A construct that specifically binds TNFα, which includes or essentially consists of an ISVD according to any of the preceding claims 1-6 or a compound according to any of claims 7-11, and which further includes one or more groups, residues, components, or linkers units optionally linked together by one or more peptide linkers, wherein said one or more groups, residues, components or linking units are selected from the group consisting of a polyethylene glycol molecule, whey proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins. 13. A composition comprising an ISVD according to any one of claims 1-4 in an active amount for use in the treatment of inflammatory bowel disease (IBD), irritable bowel syndrome, Crohn's disease, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, digestive tract injury and/ or cancer of the digestive tract. 14. A composition containing a compound according to any one of claims 6 to 11 in an active amount, for use in the treatment of inflammatory bowel disease (IBD), irritable bowel syndrome, Crohn's disease, ulcerative colitis, mucositis, aphthous stomatitis, celiac disease, trauma of the digestive tract and / or cancer of the digestive tract.

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