VH3-binding polypeptides

Abstract

The present disclosure relates to a class of engineered polypeptides that have binding affinity for the VH3 region of immunoglobulins and exhibit desirable alkaline wash stability properties. Additionally, the polypeptides exhibit significantly reduced binding affinity for the Fc region of immunoglobulins. The present disclosure also relates to methods for isolating immunoglobulins or fragments thereof using the polypeptides, as well as related products.

Description

[Technical field]

[0001] The present disclosure relates to a class of engineered polypeptides that have binding affinity for the VH3 region of immunoglobulins and exhibit desirable alkaline wash stability properties. Additionally, the polypeptides exhibit significantly reduced binding affinity for the Fc region of immunoglobulins. The present disclosure also relates to methods for isolating immunoglobulins or fragments thereof using the polypeptides, as well as related products. [Background technology]

[0002] Immunoglobulins are the most widely used biopharmaceuticals worldwide, either in production or development. Their high commercial demand for specific therapeutic markets, and therefore their value, has placed emphasis on pharmaceutical companies to maximize the productivity of their manufacturing processes for mAbs and / or their fragments, while controlling the associated costs.

[0003] Affinity chromatography is often used as one of the key steps in the purification of those immunoglobulin molecules, such as monoclonal or polyclonal antibodies, or fragments thereof. A particularly interesting class of affinity reagents are proteins that can specifically bind to the constant parts of immunoglobulin molecules, such interaction being independent of the antigen-binding specificity of the antibody. Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples, such as, but not limited to, serum or plasma preparations, or feedstocks derived from cell cultures. One example of such a protein is Staphylococcus aureus protein A (SpA), which contains domains capable of binding to the Fc and Fab proteins of IgG immunoglobulins from different species. These domains are generally designated as E, D, A, B and C domains.

[0004] Due to their high affinity and selectivity, SpA-based proteins are widely used in the field of biotechnology, for example as ligands in affinity chromatography for antibody capture and purification, as well as for detection or quantification. Currently, SpA-based affinity media are perhaps the most widely used affinity media for the isolation of monoclonal antibodies and their fragments from different samples, including industrial cell culture supernatants. Thus, various matrices containing Protein A or Protein A-derived ligands are commercially available, for example, from Cytiva™, Uppsala, Sweden, in the form of MabSelect™ SuRe, MabSelect™ SuRe LX, MabSelect™ PrismA, and HiScreen Fibro™ PrismA.

[0005] Certain Protein A and Protein A-derived ligands have binding affinity for both the Fc portion of antibodies and some VH domains of antibodies, especially VH3. As a result, co-purification of product-related impurities, such as half antibodies and truncated variants, can occur, requiring complex and / or not sufficiently gentle elution schemes. Furthermore, purification of bispecific antibodies, such as emicizumab or fragments thereof, requires complex elution schemes to ensure that only correctly paired bispecific antibodies or fragments thereof are obtained, and not a mixture of correctly and incorrectly paired bispecific antibodies and fragments. Thus, there is an unmet need in the field of simplified and reliable isolation methods for antibodies and fragments thereof. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2003080655 [Patent Document 2] International Publication No. 2008039141 [Patent Document 3] International Publication No. 2015005859 [Patent Document 4] U.S. Pat. No. 10,308,690 [Patent Document 5] International Publication No. 2016079033 [Patent Document 6] International Publication No. 2017194596 [Patent Document 7] US Patent No. 9,663,558 [Patent Document 8] International Publication No. 2016079034 [Patent Document 9] JP 2006304633 A [Patent Document 10] International Publication No. 2007097361 [Patent Document 11] European Patent Application Publication No. 1992692 [Patent Document 12] International Publication No. 2012083425 [Patent Document 13] International Publication No. 2012086660 [Patent Document 14] US Patent Publication No. 20120208234 [Patent Document 15] European Patent Application Publication No. 2495254 [Patent Document 16] International Publication No. 2013109302 [Patent Document 17] International Publication No. 2015034000 [Patent Document 18] International Publication No. 2015034056 [Patent Document 19] China Patent Application Publication No. 105481954 [Patent Document 20] International Publication No. 2016152946 [Patent Document 21] International Publication No. 2017009421 [Patent Document 22] International Publication No. 2017014261 [Patent Document 23] International Publication No. 2018009006 [Patent Document 24] International Publication No. 2018029158 [Patent Document 25] International Publication No. 2018029157 [Patent Document 26] International Publication No. 2019030156 [Patent Document 27] China Patent Application Publication No. 109721645 [Patent Document 28] International Publication No. 2019093439 [Patent Document 29] International Publication No. 2020040307 [Patent Document 30] International Publication No. 2020157281 [Patent Document 31] U.S. Patent No. 6,602,990 [Patent Document 32] U.S. Patent No. 7,396,467 [Patent Document 33] International Publication No. 2019 / 137869 [Patent Document 34] International Publication No. 2018 / 011600 [Patent Document 35] U.S. Patent No. 6,399,750 [Non-patent literature]

[0007] [Non-Patent Document 1] Sjoedahl, Eur J Biochem 1977 September;78(2):471-90 [Non-Patent Document 2] Thompson et al., Nucleic Acids Research, 22:4673-4680 (1994) [Non-Patent Document 3] Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp. 6-13 [Non-Patent Document 4] S Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964) [Non-Patent Document 5] "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988) Summary of the Invention

[0008] It is an object of the present disclosure to provide VH3-binding SpA-derived ligands that exhibit desirable alkaline wash stability properties. Such ligands can be used, for example, in methods for isolating immunoglobulins and / or fragments thereof.

[0009] It is one object of the present disclosure to provide VH3-binding SpA-derived ligands that exhibit no or significantly reduced affinity for the Fc (fragment crystallized) region of immunoglobulins.

[0010] It is an object of the present disclosure to provide VH3-binding SpA-derived ligands that allow for the efficient isolation of immunoglobulins and / or fragments thereof, and that alleviate the above and other disadvantages of the prior art.

[0011] One objective is to provide a VH3-binding SpA-derived ligand that allows gentle elution of purified antibodies.

[0012] It is a further object to provide VH3 binding SpA derived ligands that reduce or prevent product-related impurities, such as co-purification of half antibodies and truncated variants.

[0013] It is an object of the present disclosure to provide VH3-binding SpA-derived ligands that are alkaline wash stable, such that such ligands can be used repeatedly, for example, to isolate immunoglobulins and / or fragments thereof.

[0014] One objective is to provide VH3-binding SpA-derived ligands that avoid competing interactions and thus are useful in a platform solution for the purification of all VH3-containing products.

[0015] One objective is to provide VH3-binding SpA-derived ligands that exhibit increased binding affinity for VH3-containing products, in other words, that are capable of binding more VH3-containing products, since they avoid competing binding interactions with Fc.

[0016] It is a further object to provide VH3-binding SpA-derived ligands which avoid competitive interactions, for example to allow elution of asymmetric antibodies and / or fragments thereof, such as bispecific or trispecific antibodies and / or fragments thereof.

[0017] These and other objects which will be apparent to those skilled in the art from this disclosure are met by different aspects of the present invention as claimed in the appended claims and as generally disclosed herein.

[0018] Thus, in a first aspect of the disclosure there is provided a VH3 binding polypeptide or any domain thereof derived from Staphylococcus protein A (SpA), said polypeptide having binding affinity to the VH3 region of trastuzumab and having a lower binding affinity to the Fc region of trastuzumab compared to the binding affinity of SEQ ID NO: 88 to the same Fc region, said VH3 binding polypeptide being alkaline wash stable.

[0019] Alternatively, the VH3 binding polypeptides disclosed herein may be evaluated for their binding affinity to the Fc region of trastuzumab, relative to the affinity of SEQ ID NO: 89 to the same Fc region. Thus, alternatively, a VH3 binding polypeptide or any domain thereof derived from Staphylococcus protein A (SpA) is provided, said polypeptide having binding affinity to the VH3 region of trastuzumab and having a lower binding affinity to the same Fc region compared to the binding affinity of SEQ ID NO: 89 to the Fc region of trastuzumab, said VH3 binding polypeptide being alkaline wash stable. Thus, in some embodiments, it is understood that a VH3 binding polypeptide or any domain thereof derived from Staphylococcus protein A (SpA) is provided, as disclosed herein, having a lower binding affinity to the same Fc region compared to the binding affinity of SEQ ID NO: 88 and SEQ ID NO: 89 to the Fc region of trastuzumab.

[0020] The skilled artisan will appreciate that the VH3 binding polypeptides derived from Staphylococcal protein A (SpA) disclosed herein, or any domains thereof, may for example, but not necessarily, be derived from any one of domains A (SEQ ID NO: 92), B (SEQ ID NO: 91), C (SEQ ID NO: 90), D (SEQ ID NO: 93) and E (SEQ ID NO: 94) of SpA, or a derivative thereof, such as domain Z (SEQ ID NO: 89), or a variant thereof, such as SEQ ID NO: 88. Thus, a VH3 binding polypeptide derived from an SpA domain may be a derivative, mutant, variant or fragment of an SpA domain as defined above.

[0021] Thus, the VH3 binding polypeptides of the present disclosure are characterized by their retention of their inherent ability to bind to the VH3 of antibodies / immunoglobulins, in particular the VH3 region of the antibody Trastuzumab. They may also exhibit reduced or lost binding affinity to the Fc region of the antibody Trastuzumab. Those skilled in the art will appreciate that similar effects are observed for antibodies or fragments thereof that contain the same or similar VH3 region as Trastuzumab, as illustrated in the appended examples.

[0022] In the context of binding affinity to VH3, the term VH3 as used herein relates to the VH3 region of the antibody Trastuzumab (trade names Herceptin™ (Roche), Trazimera™ (Pfizer)), which region comprises the amino acid residues at the following positions according to the Kabat numbering system: H15:G; H17:S; H19:R; H57:T; H59:Y; H64:K; H65:G; H66:R; H68:T; H70:S; H81:Q; H82a:N and H82b:S. For the avoidance of any doubt, the terms "VH3 region of Trastuzumab" and "VH3" (without further specification) are used to refer to the VH3 region of the antibody Trastuzumab. The same VH3 region is also present in the following antibodies: avelumab (trade name Bavencio™), denosumab (trade name Xgeva™), dupilumab (trade name Dupixent™), and emicizumab (trade name Hemlibra™). The VH3 region of trastuzumab is contained in the fragment antigen-binding region (Fab region) of trastuzumab, the Fab being defined by SEQ ID NOs: 182 and 183.

[0023] In the context of "binding affinity to Fc", the term Fc (fragment crystallized) as used herein refers to the Fc region of the antibody Trastuzumab (trade names Herceptin™ (Roche), Trazimera™ (Pfizer)). The Fc region of Trastuzumab corresponds to the Fc region of the antibody subclasses IgG1, IgG2 and IgG4. For the avoidance of any doubt, the terms "Fc region of Trastuzumab" and "Fc" (without further elaboration) are used to refer to the Fc region of the antibody Trastuzumab. The Fc region of Trastuzumab is defined by SEQ ID NO: 184.

[0024] Furthermore, the VH3 binding polypeptides according to the present disclosure are characterized by their ability to bind to VH3, in other words their affinity for VH3, which is not affected or only slightly affected by exposure to alkaline conditions, such as, for example, the conditions under which a separation matrix containing said VH3 binding polypeptides is washed. Such conditions may be washing with 0.5 M NaOH or KOH or other suitable washing solutions known in the art. The skilled artisan will recognize the conditions conventionally used to wash separation matrices and separation columns before their repeated use. The term "alkaline washing stability" as used herein refers to the property of a VH3 binding polypeptide with respect to its affinity for VH3 after exposure to 0.5 M NaOH. A VH3 binding polypeptide disclosed herein is considered to be alkaline washing stable if it retains at least 20% of its VH3 binding ability, in other words its affinity for VH3, after 100 washing cycles compared to its VH3 binding ability / affinity for VH3 before the first washing cycle. This evaluation of alkaline washing stability may be as described in the attached Example 1.

[0025] In particular, as discussed in detail below, the VH3 binding polypeptides disclosed herein retain a significant portion of their VH3 binding properties, even after repeated alkaline wash steps.

[0026] In one embodiment of this aspect, the VH3 binding polypeptide comprises sequence A, which sequence A is: i) X8X9X 10 X 11 AX 13 X 14 X 15 X 16 X 17 X 18 X 19 PNLX 23 X 24 X 25 X 26 R X 28 X 29 FIQSLX 35 X 36 (SEQ ID NO:95) (Independently of each other, X8 is selected from E, D and A; X 15 is selected from E and Q; X 16 is selected from I and V; X 19 is selected from L and M; X 23 is selected from N and T; X 24 is selected from A and E, for example, E; X 25 is selected from D and E, for example E; X 35 is selected from K, R and H; X 36 is selected from D and H); ii) an amino acid sequence having at least 83% identity to the sequence defined under i); iii) an amino acid sequence having at least 70% identity to any sequence selected from the group consisting of residues 8 to 36 in SEQ ID NO: 88, residues 8 to 36 in SEQ ID NO: 89, residues 8 to 36 in SEQ ID NO: 90, residues 8 to 36 in SEQ ID NO: 91, residues 8 to 36 in SEQ ID NO: 92, residues 11 to 39 in SEQ ID NO: 93, and residues 1 to 29 in SEQ ID NO: 94. and wherein the amino acid sequence is selected from i), ii), and iii) defined as follows: Further, in each of i), ii) and iii), independently of one another, X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11 is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G.

[0027] In one embodiment, a VH3 binding polypeptide is provided, said polypeptide having binding affinity to the VH3 region of an antibody and low binding affinity to the Fc region of an antibody, said VH3 binding polypeptide being alkaline washing stable.

[0028] The inventors have surprisingly found that VH3 binding polypeptides derived from SpA or domains thereof having the amino acid residues disclosed herein have at least the amino acid residues at positions X9, X 10 Place, X 11 Place, X 13 Rank and X 14 In the position, and optionally X 17 Place, X 18 Rank and X 26 It has been found that VH3 binding polypeptides containing amino acids defined herein at the aforementioned positions at least retain or exhibit the property of improved alkaline wash stability. Their binding affinity to the Fc of trastuzumab is also significantly reduced or eliminated. VH3 binding polypeptides containing amino acids defined herein at the aforementioned positions lose at least a significant portion of their affinity to the Fc, while retaining affinity to the VH3 region.

[0029] In one particular embodiment, the VH3 binding polypeptide has a binding affinity to the VH3 region of trastuzumab. In one particular embodiment, the VH3 binding polypeptide has a binding affinity to the VH3 region of trastuzumab, and has a lower binding affinity to the Fc region of trastuzumab than SEQ ID NO: 88 and / or SEQ ID NO: 89 have to the same region. As explained above, the VH3 binding polypeptides disclosed herein can be evaluated for their binding affinity to the Fc region of trastuzumab in comparison with the affinity of SEQ ID NO: 88 to the same Fc region. Thus, in one embodiment, a VH3 binding polypeptide is provided comprising sequence A, which consists of an amino acid sequence selected from i), ii) and iii) as defined above, and has a lower binding affinity to the Fc region of trastuzumab than SEQ ID NO: 88 has to the same Fc region. Alternatively, there is provided a VH3 binding polypeptide comprising sequence A, which consists of an amino acid sequence selected from i), ii) and iii) as defined above, and has a lower binding affinity to the Fc region of trastuzumab than the binding affinity of SEQ ID NO: 89 to the same Fc region. In some embodiments, there is provided a VH3 binding polypeptide as disclosed herein, which has a lower binding affinity to the Fc region of trastuzumab compared to the binding affinity of SEQ ID NO: 88 and SEQ ID NO: 89 to the same Fc region of trastuzumab. Thus, it will be appreciated by those skilled in the art that a comparison of the binding affinity of the VH3 binding polypeptide of the present disclosure to the Fc region of trastuzumab can be made using SEQ ID NO: 88 and / or SEQ ID NO: 89 as a control.

[0030] The inventors have surprisingly found that position X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26It has been found that by varying the amino acid residues at the positions corresponding to the positions as disclosed herein relative to a VH3 binding polypeptide or domain thereof derived from SpA, a VH3 binding polypeptide with desirable properties can be obtained. In particular, the binding affinity to the Fc region of trastuzumab is significantly reduced or eliminated, while the VH3 binding polypeptide at least retains or exhibits the property of improved alkaline wash stability. A VH3 binding polypeptide containing the amino acids defined herein at the aforementioned positions loses at least a significant part of its affinity to Fc, while retaining its affinity to the VH3 region.

[0031] In particular, in one embodiment there is provided a VH3 binding polypeptide as disclosed herein, comprising, independently of each other, in sequence i): X8 is selected from E, D and A; X 15 is selected from E and Q; X 16 is selected from I and V; X 19 is selected from L and M; X 24 is E; X 25 is E; X 23 is selected from N and T; X 35 is selected from K, R and H; and X 36 is selected from D and H.

[0032] For clarity, the percent identity in ii) and iii) is X9, X 10 , X 11 , X 13 , X 14 , X 17 , X 18 and X 26. It is understood that this does not apply to X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18Rank and X 26. The amino acid residues at positions 2) and 3) are included in the definition according to ii) and iii) as defined above in a VH3 binding polypeptide.

[0033] The above definition of the classes of sequence-related VH3 binding polypeptides is based on the analysis of several VH3 binding polypeptide variants selected for the properties of at least maintained affinity for VH3 and alkaline wash stability, as described in the appended Examples. Also, affinity to Fc was evaluated, and VH3s were selected for reduced or lost affinity for Fc.

[0034] As described herein, the affinity of trastuzumab to VH3 and Fc was evaluated. As described above, trastuzumab can be defined by the amino acid residues at the aforementioned positions according to the Kabat numbering system. Trastuzumab can be defined as having a heavy chain amino acid sequence according to SEQ ID NO: 183 and a light chain amino acid sequence according to SEQ ID NO: 184.

[0035] The VH3 binding polypeptide may comprise the identified sequence A, which corresponds to the region of the parent scaffold that interacts and binds with Fc. This region comprises two alpha helices, helix 1 and helix 2, within a three-helix bundle protein domain. Helix 2 and helix 3 of this three-helix bundle protein comprise the binding surface for interaction with the VH3 region of an antibody.

[0036] As those skilled in the art will recognize, the function of any polypeptide depends on the tertiary structure of that polypeptide. Therefore, it is possible to make minor changes to the sequence of amino acids in a polypeptide without affecting its function. Thus, the present disclosure includes modified variants of VH3 binding polypeptides. These modified variants are alkaline wash stable. The modified variants are those in which their affinity to the VH3 region is at least retained. The variants are those in which their affinity to the Fc region is significantly reduced or eliminated when compared to their affinity to the VH3 region.

[0037] The modifications of Fc binding affinity identified by the inventors are applicable to VH3 binding polypeptides based on or derived from different three-helix domains of protein A from Staphylococcus aureus, such as any one of domains A (SEQ ID NO: 92), B (SEQ ID NO: 91), C (SEQ ID NO: 90), D (SEQ ID NO: 93) and E (SEQ ID NO: 94), in particular domain B, and derivatives thereof. In particular, said modifications are applicable to VH3 binding polypeptides based on or derived from the three-helix bundle protein domain Z from domain B of staphylococcal protein A (SEQ ID NO: 89), or to variants of SEQ ID NO: 88.

[0038] Such VH3 binding polypeptides are described in WO 2003080655, Sjoedahl, Eur J Biochem September 1977; 78(2): pp. 471-90, International Publication No. 2008039141, International Publication No. 2015005859, U.S. Patent No. 10308690, International Publication No. 2016079033, International Publication No. 2017194596, U.S. Patent No. 9663558, International Publication No. 2016079034, JP 2006304633 A, International Publication No. 2007097361, European Patent Application Publication No. 1992692, International Publication No. 2012083425, International Publication No. 2012086660, U.S. Patent Application Publication No. 20120208234, European Patent Application Publication No. 249 No. 5254, International Publication No. 2013109302, International Publication No. 2015034000, International Publication No. 2015034056, Chinese Patent Application Publication No. 105481954, International Publication No. 2016152946, International Publication No. 2017009421, International Publication No. 2017014261, International Publication No. 2018009006, International Publication No. 2018029158, International Publication No. 2018029157, International Publication No. 2019030156, Chinese Patent Application Publication No. 109721645, International Publication No. 2019093439, International Publication No. 2020040307, ​​and International Publication No. 2020157281.

[0039] Therefore, the inventors have proposed a method for the preparation of a nucleotide sequence at positions X9, X10, and X20 disclosed herein to achieve the effect of significantly reducing or eliminating binding affinity to Fc while retaining the VH3 binding characteristics and at least the alkaline wash stability properties. 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26 It is envisaged that all, or a subset, of the amino acid residues at positions may be introduced into any one of the previously known VH3 binding polypeptides.

[0040] Also included in the disclosure is a VH3 binding polypeptide comprising a sequence A having at least 83% identity to a polypeptide defined in i). In some embodiments, the polypeptide may comprise a sequence that is at least 87%, such as at least 90%, such as at least 93%, such as at least 96% identical to a polypeptide defined in i). For example, it is possible that one amino acid residue belonging to a particular functional group of amino acid residues (e.g., hydrophobic, hydrophilic, polar, etc.) may be replaced with another amino acid residue from the same functional group. In some embodiments, such changes are at positions X9, X10, X111, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X30, X31, X32, X33, X34, X35, X36, X37, X38, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, X50, X51, X52, X53, X54, X55, X56, X57, X60, X61, X62, X63, X64, X65, X70, X71, X72, X83, X90, X91, X92, X93, X94, X95, X96, X97, X98, X99, X109, X110, X121, X130, X142, X153, X165, X176, X189, X197, X198, X199, X199, X200, X201, X202, X203, X204, X205, X206, X207, X208, X210, X211, X2 10 , X 11 , X 13 , X 14 , X 17 , X 18 and X 26 In other embodiments, such changes may be made only at non-variable positions, also designated scaffold amino acid residues. In such cases, no changes are allowed at the variable positions, i.e., positions designated by "X" in sequence i).

[0041] Also included in the disclosure is a VH3 binding polypeptide comprising sequence A, wherein sequence A has at least 70% identity to any sequence selected from the group consisting of residues 8-36 in SEQ ID NO:88, residues 8-36 in SEQ ID NO:89, residues 8-36 in SEQ ID NO:90, residues 8-36 in SEQ ID NO:91, residues 8-36 in SEQ ID NO:92, residues 11-39 in SEQ ID NO:93, and residues 1-29 in SEQ ID NO:94. In some embodiments, the polypeptide may comprise a sequence that is at least 74%, such as at least 77%, such as at least 80%, such as at least 83%, such as at least 87%, such as at least 90%, such as at least 93%, such as at least 96% identical to any sequence selected from the group consisting of residues 8-36 in SEQ ID NO:88, residues 8-36 in SEQ ID NO:89, residues 8-36 in SEQ ID NO:90, residues 8-36 in SEQ ID NO:91, residues 8-36 in SEQ ID NO:92, residues 11-39 in SEQ ID NO:93, and residues 1-29 in SEQ ID NO:94. In certain embodiments, the sequence is selected from the group consisting of residues 8-36 in SEQ ID NO:88, residues 8-36 in SEQ ID NO:89, residues 8-36 in SEQ ID NO:90, residues 8-36 in SEQ ID NO:91, residues 8-36 in SEQ ID NO:92, residues 11-39 in SEQ ID NO:93, and residues 1-29 in SEQ ID NO:94. In certain embodiments, the sequence is selected from the group consisting of residues 8-36 in SEQ ID NO:88 and residues 8-36 in SEQ ID NO:89. In one embodiment, the sequence is SEQ ID NO:88. In one embodiment, the sequence is SEQ ID NO:89.

[0042] The term "% identity" as used throughout this disclosure may be calculated, for example, as follows: The query sequence is aligned with the target sequence using the CLUSTAL W algorithm (Thompson et al., Nucleic Acids Research, 22:4673-4680 (1994)). Comparison is made over a window corresponding to the shortest sequence among the aligned sequences. The shortest sequence among the aligned sequences may in some cases be the target sequence. In other cases, the query sequence may constitute the shortest sequence among the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have an identical counterpart in the target sequence is reported as % identity. In one embodiment of the VH3 binding polypeptide disclosed herein, the sequence A satisfies criterion i) and criterion ii), or criteria i) and iii), or criteria ii) and iii). In one embodiment, the sequence A satisfies all criteria i), ii), and iii).

[0043] Any VH3 binding polypeptide, including but not limited to, position X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26Also included in the present disclosure are polypeptides derived from Staphylococcus aureus protein A (SpA) or any domain thereof, comprising an amino acid residue as defined herein at a position corresponding to the amino acid residues at positions ...

[0044] In particular, the present disclosure includes any VH3 binding polypeptide, including, but not limited to, any of the VH3 binding polypeptides at position X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26 and wherein the VH3 binding polypeptide exhibits the properties of reduced or abolished affinity for the Fc of trastuzumab compared to SEQ ID NO: 88, affinity for the VH3 of trastuzumab compared to SEQ ID NO: 88, and is alkaline wash stable. In this context, the term "corresponding position" as used herein refers to the amino acid residues X9, X10, X11, X20, X30, X40, X50, X60, X70, X80, X90, X100, X111, X21, X32, X43, X54, X65, X76, X86, X90, X102, X113, X125, X134, X140, X156, X166, X170, X187, X190, X225, X230, X240, X256, X350, X360, X370, X440, X450, X560, X670, X787, X890, X90, X191, X192, X193, X254, X370, X455, X560, X687, X790, X890, X90, X194, X195, X260, X370, X455, X560, X687, X790, X890, X ... 10 , X 11 , X 13 , X 14 , X 17 , X 18 and X 26The presence or absence of amino acid residues X9, X 10 , X 11 , X 13 , X 14 , X 17 , X 18 and X 26 and / or do not significantly alter the alpha helical tertiary structure in the region of the VH3 binding polypeptide comprising said residues and / or do not alter the VH3 binding surface comprising said residues. 10 , X 11 , X 13 , X 14 , X 17 , X 18 and X 26 It should be understood that the presence or absence of an amino acid residue between positions X9, X8, and X9 in SEQ ID NO: 88 should not be construed as meaning that said position does not correspond to a position of SEQ ID NO: 88. Instead, one or more deletion or insertion mutations may be made between amino acid residues X9, X10, and X11 in SEQ ID NO: 88, which mutations do not significantly alter the alpha helical tertiary structure and / or the VH3 binding surface of the region of the VH3 polypeptide that includes said residues. 10 , X 11 , X 13 , X 14 , X 17 , X 18 and X 26 Also encompassed herein are any VH3 binding polypeptides comprising between positions corresponding to:

[0045] Thus, in one embodiment there is provided a VH3 binding polypeptide or any domain thereof derived from Staphylococcus aureus protein A (SpA), comprising at position X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26 The positions in the alignment with SEQ ID NO: 88 corresponding to the positions are, independently of one another, as follows: X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G.

[0046] The skilled person will understand that said alignment may equally be performed with SEQ ID NO: 89. Thus, in another embodiment, there is provided a VH3 binding polypeptide or any domain thereof derived from Staphylococcus aureus protein A (SpA), comprising: 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26 The positions in the alignment with SEQ ID NO: 89 corresponding to the positions are, independently of one another, as follows: X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11 is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G.

[0047] In one embodiment according to the first aspect disclosed herein, X9 is selected from Q and Y. In another embodiment, X9 is selected from Q and A. In one embodiment, X9 is selected from A and Y. In one embodiment, X9 is A. In one embodiment, X9 is Y. In one embodiment, X9 is Q.

[0048] In one embodiment, X 10 is Q. In one embodiment, X 10 is Y.

[0049] In one embodiment, X 11 is selected from T and E. In one embodiment, X 11 is selected from T and R. In one embodiment, X 11 is selected from R and E. In one embodiment, X 11 is T. In one embodiment, X 11 is E. In one embodiment, X 11 is R.

[0050] In one embodiment, X 13 is selected from L, E, R and A; or from L, E, R and Q; or from L, E, A and Q; or from L, R, A and Q; or from E, R, A and Q; in particular from L, E, R and A; or from L, E, R and Q; or from L, E, A and Q; or from L, R, A and Q. In one embodiment, X 13 are selected from L, E and R; or from L, E and A; or from L, E and Q; or from L, R and A; or from L, R and Q; or from L, A and Q; or from E, R and A; or from E, R and Q; or from E, A and Q; or from R, A and Q; in particular selected from L, E and R; or from L, E and A; or from L, E and Q; or from L, R and A; or from L, R and Q. In one embodiment, X 13is selected from L and E; or from L and R; or from L and A; or from L and Q; or from E and R; or from E and A; or from E and Q; or from R and A; or from R and Q; or from A and Q; in particular from L and E; or from L and R; or from L and A; or from L and Q. In one embodiment, X 13 is L. In one embodiment, X 13 is A. In one embodiment, X 13 is E. In one embodiment, X 13 is Q. In one embodiment, X 13 is R.

[0051] In one embodiment, X 14 is selected from A, W, E, L and Q; or from A, W, E, L and R; or from A, W, E, Q and R; or from A, W, L, Q and R; or from A, E, L, Q and R; or from W, E, L, Q and R. In one embodiment, X 14 are selected from A, W, E and L; or from A, W, E and Q; or from A, W, E and R; or from A, W, L and Q; or from A, W, L and R; or from A, W, Q and R; or from A, E, L and Q; or from A, E, L and R; or from A, E, Q and R; or from A, L, Q and R; or from W, E, L and Q; or from W, E, L and R; or from W, E, Q and R; or from W, L, Q and R; or from E, L, Q and R. In one embodiment, X 14 are selected from A, W and E; or from A, W and L; or from A, W and Q; or from A, W and R; or from A, E and L; or from A, E and Q; or from A, E and R; or from A, L and Q; or from A, L and R; or from A, Q and R; or from W, E and L; or from W, E and Q; or from W, E and R; or from W, L and Q; or from W, L and R; or from W, Q and R; or from E, L and Q; or from E, L and R; or from E, Q and R; or from L, Q and R. In one embodiment, X 14is selected from A and W; or from A and E; or from A and L; or from A and Q; or from A and R; or from W and E; or from W and L; or from W and Q; or from W and R; or from E and L; or from E and Q; or from E and R; or from L and Q; or from L and R; or from Q and R. In one embodiment, X 14 is A. In one embodiment, X 14 is W. In one embodiment, X 14 is E. In one embodiment, X 14 is L. In one embodiment, X 14 is Q. In one embodiment, X 14 is R.

[0052] In one embodiment, X 17 is selected from A and H. In one embodiment, X 17 is selected from A and L. In one embodiment, X 17 is selected from H and L. In one embodiment, X 17 is A. In one embodiment, X 17 is H. In one embodiment, X 17 is L.

[0053] In one embodiment, X 18 is selected from R and L. In one embodiment, X 18 is selected from R and H. In one embodiment, X 18 is selected from H and L. In one embodiment, X 18 is R. In one embodiment, X 18 is L. In one embodiment, X 18 is H.

[0054] In one embodiment, X 26 is Q. In one embodiment, X 26 is S.

[0055] In one embodiment, X 28 is N. In one embodiment, X 28 is A.

[0056] In one embodiment, X 29 is A. In one embodiment, X 29 is G.

[0057] In one more specific embodiment defining a subclass of the VH3 binding polypeptides disclosed herein, X 14 If A, then X 10 is Y or X9X 10 is AY. X 14 is A and X 10 Particular examples of VH3 binding polypeptides in which X is Y are SEQ ID NOs: 2, 7, 10, 11, 13, 14, 15, 16, 21, 27, 31, 36, 41, 43, 48, 51, 52, 54, 55, 56, 57, 62, 67, 72, 77, 82, 83, 84, 85, 86 and 87. 14 is A and X9X 10 Particular examples of VH3 binding polypeptides in which is AY are SEQ ID NOs: 7, 10, 11, 13, 14, 15, 16, 21, 27, 31, 36, 41, 48, 51, 52, 54, 55, 56, 57, 62, 67, 72, 77, 82, 83, 84, 85, 86 and 87. Similarly, by studying the sequences of the VH3 binding polypeptides shown in Figure 5, it is easy to identify which VH3 binding polypeptides are included in each embodiment disclosed herein.

[0058] Other embodiments defining subclasses of the VH3 binding polypeptides disclosed herein may be as follows.

[0059] In one specific embodiment, which defines a subclass of the VH3 binding polypeptides disclosed herein, X9X 10 X 11 is selected from the group consisting of QQT, QYT, YQT, AQE, AQR and AYR; for example, the group consisting of QQT, QYT, AQE, AQR and AYR; for example, the group consisting of AQE, AQR and AYR; for example, the group consisting of AQR and AYR, for example, X9X 10 X 11 In another embodiment, X is AYR. 17 X 18is selected from the group consisting of AR, HL and LH; for example, from the group consisting of AR and HL or from the group consisting of AR and LH; for example, X 17 X 18 In one embodiment, X is AR. 24 X 25 is EE. In one embodiment, X 28 X 29 is selected from the group consisting of NA, NG, AA and AG; for example, the group consisting of NA, NG and AG or the group consisting of NG, AA and AS; for example, the group consisting of NG and AG or the group consisting of AG and NA or the group consisting of AG and AA. In one embodiment, X 28 X 29 is AA. In one embodiment, X 28 X 29 is AG. In one embodiment, X 28 X 29 is NG. In one embodiment, X 28 X 29 In one embodiment, independently of each other, X9X 10 X 11 is selected from the group consisting of AQE, AQR and AYR; X 13 X 14 are selected from the group consisting of LA, LW, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ and RR; for example, independently of each other, XX 10 X 11 is selected from the group consisting of AQE, AQR and AYR; X 13 X 14 are selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ and RR; for example, independently of each other, XX 10 X 11 is selected from the group consisting of AQR and AYR; 13 X 14is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ and RR; for example, XX 10 X 11 is AYR, and X 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, and RR. 10 X 11 X 13 X 14 is selected from the group consisting of QQTLA, QYTLA, YQTLA, AQELA, AQRLA, AYRLA and AYRLW; for example, the group consisting of QQTLA, QYTLA, AQELA, AQRLA, AYRLA and AYRLW; for example, the group consisting of AQELA, AQRLA, AYRLA and AYRLW; for example, the group consisting of AQRLA and AYRLA; for example, X9X 10 X 11 X 13 X 14 is AYRLA.

[0060] In one embodiment, independently of each other, X9X 10 X 11 is selected from the group consisting of AQE, AQR and AYR; X 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR and LW; 17 X 18 are selected from LH and AR; for example, independently of each other, XX 10 X 11 is selected from the group consisting of AQR and AYR; 13 X 14is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR and LW; 17 X 18 are selected from LH and AR; for example, independently of each other, XX 10 X 11 is AYR, and X 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR and LW; 17 X 18 are selected from LH and AR; for example, independently of each other, XX 10 X 11 is AYR, and X 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR and LW; 17 X 18 is AR.

[0061] In one embodiment, X9X 10 X 11 X 13 X 14 X 17 X 18is selected from the group consisting of QQTLALH, QYTLALH, YQTLALH, QQTLAAR, AQELALH, AQRLALH, AYRLALH, AYRLWLH, AYRLWAR, AYRLAAR, AYRLAHL and AYRLWHL; for example, the group consisting of QQTLALH, QYTLALH, QQTLAAR, AQELALH, AQRLALH, AYRLALH, AYRLWLH, AYRLWAR, AYRLAAR, AYRLAHL and AYRLWHL; for example, the group consisting of AQELALH, AQRLALH, AYRLALH, AYRLWAR, AYRLAAR and AYRLAHL; for example, the group consisting of AQRLALH, AYRLALH and AYRLAAR; for example, the group consisting of AQRLALH and AYRLAAR; for example, X9X 10 X 11 X 13 X 14 X 17 X 18 In one embodiment, X is AYRLAAR. 24 X 25 X 28 X 29 is selected from the group consisting of EENA, EENG, EEAA and EEAG; for example, the group consisting of EENA, EENG and EEAG; for example, the group consisting of EENG and EEAG; for example, X 24 X 25 X 28 X 29 is EEAG.

[0062] In one embodiment, X 26 X 28 X 29 is selected from the group consisting of QNG, QAA, QAG, QNA and SAG; for example, the group consisting of QNG, QAG, QNA and SAG; for example, the group consisting of QNG, QAG and SAG and / or the group consisting of QNG, QAG, QNA or the group consisting of QNG, QNA and SAG; for example, the group consisting of QAG and SAG; for example, X 26 X 28 X 29 is QAG or SAG.

[0063] In one embodiment, X 26 X 28X 29 is selected from the group consisting of QAG and SAG; for example, X 26 X 28 X 29 is QAG or SAG. As described in detail in the subsequent experimental section, the selection of VH3-binding polypeptide variants led to the identification of several individual amino acid sequences according to sequence A as defined herein, which sequences constitute individual embodiments of sequence i) according to this aspect. The sequences of the individual amino acid sequences according to sequence A correspond to amino acids 8 to 36 in SEQ ID NOs: 1 to 82 as presented in FIG. 5.

[0064] Thus, in one embodiment according to the first aspect disclosed herein, there is provided a VH3-binding polypeptide in which sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, for example, from the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, for example, from the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56. In one embodiment, sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 41, for example, from the group consisting of SEQ ID NOs: 1 to 40, for example, from the group consisting of SEQ ID NOs: 1 to 15. In one embodiment, sequence i) corresponds to a sequence from 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54, and 56 to 81; for example, the group consisting of SEQ ID NOs: 13, 15, 54, and 56. In one embodiment, sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82, for example, from the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or from the group consisting of SEQ ID NOs: 15, 41, 56 and 82. In one embodiment, sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56. In one embodiment, sequence i) corresponds to a sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15.In one embodiment, sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 15 to 41, for example, from the group consisting of SEQ ID NOs: 15 to 40 or from the group consisting of SEQ ID NOs: 15 and 41. In one embodiment, sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 14 and 15.

[0065] In one embodiment, sequence i) corresponds to a sequence from 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 42, 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54, 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56 and 56. In one embodiment, sequence i) corresponds to a sequence from 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 56 to 82, for example, the group consisting of SEQ ID NOs: 56 to 81, or the group consisting of SEQ ID NOs: 56 and 82. In one embodiment, sequence i) corresponds to a sequence from 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 55 and 56.

[0066] In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 13. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 14. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 15. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 41. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 54. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 55. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 56. In one embodiment, sequence i) corresponds to the sequence of positions 8 to 36 in SEQ ID NO: 82.

[0067] In some embodiments of the present disclosure, the VH3 binding polypeptide is based on a variant of the SpA domain having mutations according to the present disclosure. Thus, in one particular embodiment, there is provided a VH3 binding polypeptide as disclosed herein having a sequence selected from the group consisting of: a sequence corresponding to residues 8-36 in SEQ ID NO: 83; a sequence corresponding to residues 8-36 in SEQ ID NO: 84; a sequence corresponding to residues 8-36 in SEQ ID NO: 85; a sequence corresponding to residues 11-39 in SEQ ID NO: 86; and a sequence corresponding to residues 1-29 in SEQ ID NO: 87.

[0068] In some embodiments of the present disclosure, the polypeptide or sequence A derived from SpA, as defined above, "forms part of" a three-helix bundle protein domain. This is understood to mean that the sequence of the polypeptide or sequence A derived from SpA is "inserted" or "grafted" into the sequence of the original three-helix bundle domain, such that the grafted sequence replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, sequence A is believed to constitute two of the three helices of the three-helix bundle and may therefore replace such a two-helix motif in any three-helix bundle. As the skilled person will recognize, the replacement of two helices of the three-helix bundle domain by two sequences A must be done so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Cα backbone of the polypeptide according to this embodiment of the present invention is substantially the same as the folding of the three-helix bundle protein domain of which it forms part, e.g. with the same elements of secondary structure in the same order. Thus, sequence A according to the present disclosure "forms part of" a three-helix bundle domain, implying that if the polypeptide according to this embodiment has the same fold as the original domain, basic structural properties are shared, such as properties that result in similar CD spectra. The skilled artisan will know other parameters that are relevant.

[0069] Thus, in one embodiment, the polypeptide or sequence A from SpA forms part of a three-helix bundle protein domain. In some embodiments, said sequence A forms part of, or essentially forms, two helices, including an interconnecting loop, within said three-helix bundle protein domain. In particular, said three-helix bundle protein domain may be selected from a domain from a bacterial receptor protein, in other words a bacterial receptor domain. Non-limiting examples of such domains are the five different three-helix domains of protein A from Staphylococcus aureus, such as domains A, B, C, D and E, in particular domain B and its derivatives, and domain C and its derivatives. An example of a derivative of said domain B is domain Z. Another example of a derivative of domain C is domain C with the mutation G29A. In one embodiment, said three-helix bundle protein domain is selected from a domain of protein A from Staphylococcus aureus or a derivative thereof. In some embodiments, the three-helix bundle protein domain is a variant of domain Z from domain B of staphylococcal protein A. In some embodiments, the three-helix bundle protein domain is domain C that contains the mutation G29A.

[0070] The skilled artisan will appreciate that the third helix of a three-helix bundle may be provided in a VH3 binding polypeptide as defined herein, and that the amino acid sequence of the third helix may vary without significantly deviating from or significantly altering the secondary structure of a polypeptide comprising sequence A and the third helix. Thus, in one embodiment, there is provided a VH3 binding polypeptide as defined herein, further comprising sequence B arranged at the C-terminus of said sequence A, wherein sequence B consists of an amino acid sequence selected from iv) and v), wherein iv) and v) are selected from the following: iv) DPSX 40 SX 42 X 43 X 44 LX 46 EAX 49 X 50 LNX 53 X 54(SEQ ID NO:96) (Independently of each other, X 40 is selected from Q, T and V; X 42 is selected from A, K, L and T; X 43 is selected from N, E, A and S; X 44 is selected from L, I, and V; X 46 is selected from A, G, S and K, for example, A, S and K; X 49 is selected from K and Q, for example, K; X 50 is selected from K and R; X 53 is selected from D, E and K; and X 54 is selected from A and S); v) an amino acid sequence having at least 75% identity to the sequence defined in iv); It is defined as follows:

[0071] In certain embodiments, sequence B consists of amino acid sequences selected from iv) and v), wherein in iv), independently of each other: X 40 is selected from Q, T and V; X 42 is selected from A, K, L and T; X 43 is selected from N, E, A and S; X 44 is selected from L, I, and V; X 46 is selected from A, S and K; X 49 is K; X 50 is selected from K and R; X 53 is selected from D, E and K; and X 54 is selected from A and S.

[0072] Thus, in yet another embodiment there is provided a VH3 binding polypeptide as defined herein comprising a link module sequence C, which sequence C has, from N-terminus to C-terminus, the following order: [Array A]-[Array B] with sequence A as defined herein and sequence B as defined herein, or comprises or consists of any amino acid sequence having at least 70% identity to any sequence selected from the group consisting of residues 8 to 54 in SEQ ID NO: 88, residues 8 to 54 in SEQ ID NO: 89, residues 8 to 54 in SEQ ID NO: 90, residues 8 to 54 in SEQ ID NO: 91, residues 8 to 54 in SEQ ID NO: 92, residues 11 to 57 in SEQ ID NO: 93, and residues 1 to 47 in SEQ ID NO: 94. Thus, in one embodiment, the sequence C is selected from the group consisting of amino acid sequences having at least 70% identity to any sequence selected from the group consisting of residues 8 to 54 in SEQ ID NO: 88, residues 8 to 54 in SEQ ID NO: 89, residues 8 to 54 in SEQ ID NO: 90, residues 8 to 54 in SEQ ID NO: 91, residues 8 to 54 in SEQ ID NO: 92, residues 11 to 57 in SEQ ID NO: 93, and residues 1 to 47 in SEQ ID NO: 94. In one embodiment, sequence C is selected from the group consisting of residues 8 to 54 in SEQ ID NO: 88, residues 8 to 54 in SEQ ID NO: 89, residues 8 to 54 in SEQ ID NO: 90, residues 8 to 54 in SEQ ID NO: 91, residues 8 to 54 in SEQ ID NO: 92, residues 11 to 57 in SEQ ID NO: 93 and residues 1 to 47 in SEQ ID NO: 94, each independently: X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11 is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G (The numbering of the amino acid residues corresponds to their positions in SEQ ID NO:88).

[0073] In one embodiment, the sequence C has at least 70% identity to any sequence selected from the group consisting of residues 8 to 54 in SEQ ID NO: 88, residues 8 to 54 in SEQ ID NO: 89, residues 8 to 54 in SEQ ID NO: 90, residues 8 to 54 in SEQ ID NO: 91, residues 8 to 54 in SEQ ID NO: 92, residues 11 to 57 in SEQ ID NO: 93, and residues 1 to 47 in SEQ ID NO: 94, and is at least 70% identical to any sequence selected from the group consisting of residues 8 to 54 in SEQ ID NO: 88, residues 8 to 54 in SEQ ID NO: 89, residues 8 to 54 in SEQ ID NO: 90, residues 8 to 54 in SEQ ID NO: 91, residues 11 to 57 in SEQ ID NO: 93, and residues 1 to 47 in SEQ ID NO: 94. 93, residues 11-57 in SEQ ID NO: 93, and residues 1-47 in SEQ ID NO: 94. As mentioned above, the % identity is expressed as the identity at positions X9, X10, X111, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X30, X31, X32, X33, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, X50, X51, X52, X53, X54, X55, X56, X57, X60, X61, X70, X71, X82, X92, X93, X104, X115, X126, X138, X142, X156, X166, X178, X186, X199, X201, X212, X222, X236, X244, X257, X316, X326, X45, X58, X617, X72, X83, X94, X95, X96, X107, X115, X128, X139, X259, X329, X46, X47, X59, X62, X73, X74, X85, X96, X97, X98, X109, X1109, X121, X139, X142, X157, X161, X175, X186, X199, X201, X212, X222, X236, X243, X25 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Place, X 26 Place, X 28 Rank and X 29 The term does not apply to the amino acid residue at position A, which is as defined above.

[0074] As discussed above, in some embodiments in which the VH3 binding polypeptide disclosed herein forms part of a three-helix bundle protein domain, the Fc binding polypeptide essentially forms part of at least three helices and the interconnecting loop between helix 1 (H1) and helix 2 (H2) (referred to as loop 1 (L1)), and the interconnecting loop between helix 2 (H2) and helix 3 (H3) (referred to as loop 1 (L2)). As used herein, the link module sequence C essentially forms part of at least three helices and the interconnecting loop between helix 1 and helix 2 (referred to as loop 1 (L1)), and the interconnecting loop between helix 2 and helix 3 (referred to as loop 2 (L2)). It is understood that the length of L1 and L2 may vary, for example by the addition or removal of one or more additional amino acid residues, as long as the variation does not significantly alter the three-dimensional structure of the polypeptide comprising the link module sequence C.

[0075] As explained above, sequence B comprises L2 and H3. The skilled artisan will appreciate that the amino acid sequences of L2 and H3 can be combined independently while maintaining the three-dimensional structure of the VH3-binding polypeptide. Thus, in one embodiment, there is provided a VH3-binding polypeptide as defined herein comprising a link module sequence C, which sequence C consists of the sequences [sequence A], [L2] and [H3] in the following order from N-terminus to C-terminus: [sequence A]-[L2]-[H3]; [Sequence A] is as defined herein, and independently of each other, [L2] is DPSV (SEQ ID NO:97); DPSQ (SEQ ID NO:98); EPSQ (SEQ ID NO:99); DPST(SEQ ID NO:100); EPSV (SEQ ID NO:101); and DPSL (SEQ ID NO: 102); selected from the group consisting of; [H3] SANLLAEAKKLNDA (SEQ ID NO: 103); SKEILAEAKKLNDA (SEQ ID NO: 104); SKAILAEAKKLNDA (SEQ ID NO: 105); SANLLAEAKKLNDA (SEQ ID NO: 106); SANLLAEAKKLNES (SEQ ID NO: 107); STNVLGEAKKLNES (SEQ ID NO: 108); SANVLGEAQKLNDS (SEQ ID NO: 109); SLEILCEAKKLNDA (SEQ ID NO:110); SLEILAEAKKLNDA (SEQ ID NO: 111); and SKKILKEAKKLNKA (SEQ ID NO: 112) is selected from the group consisting of:

[0076] In one embodiment, [L2] is selected from the group consisting of DPSV (SEQ ID NO: 97); DPSQ (SEQ ID NO: 98); DPST (SEQ ID NO: 100); and DPSL (SEQ ID NO: 102). In one embodiment, [L2] is selected from the group consisting of DPSV (SEQ ID NO: 97); DPSQ (SEQ ID NO: 98); and DPSL (SEQ ID NO: 102). In one embodiment, [L2] is selected from the group consisting of DPSV (SEQ ID NO: 97) and DPSL (SEQ ID NO: 102).

[0077] In one embodiment, [L2] comprises or consists of DPSV (SEQ ID NO: 97). In one embodiment, [L2] comprises or consists of DPSQ (SEQ ID NO: 98). In one embodiment, [L2] comprises or consists of DPSL (SEQ ID NO: 102). In one particular embodiment, [H3] is selected from the group consisting of SANLLAEAKKLNDA (SEQ ID NO: 103); SKEILEAKKLNDA (SEQ ID NO: 104); SKAILEAKKLNDA (SEQ ID NO: 105); SANLLAEAKKLNDA (SEQ ID NO: 106); SANLLAEAKKLNES (SEQ ID NO: 107); STNVLGEAKKLNES (SEQ ID NO: 108) and SANVLGEAQKLNDS (SEQ ID NO: 109). In one embodiment, [H3] is selected from the group consisting of SANLLAEAKKLNDA (SEQ ID NO: 103), SKEILEAKKLNDA (SEQ ID NO: 104) and SKAILEAKKLNDA (SEQ ID NO: 105), for example from the group consisting of SKEILEAKKLNDA (SEQ ID NO: 104) and SKAILEAKKLNDA (SEQ ID NO: 105), or from the group consisting of SANLLAEAKKLNDA (SEQ ID NO: 103) and SKAILEAKKLNDA (SEQ ID NO: 105), or SANLLAEAKKLNDA (SEQ ID NO: 103) and SKEILAEAKKLNDA (SEQ ID NO: 104).

[0078] In one embodiment, [H3] comprises or consists of SANLLAEAKKLNDA (SEQ ID NO: 103). In one embodiment, [H3] comprises or consists of SKEILAAKKLNDA (SEQ ID NO: 104). In one embodiment, [H3] comprises or consists of SKAILAAKKLNDA (SEQ ID NO: 105).

[0079] In one embodiment, [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); [Sequence A]-DPSQSANLLAEAKKLNES (SEQ ID NO: 116); [Sequence A]-DPSVSLEILGEAKKLNDA (SEQ ID NO: 117); [Sequence A]-DPSVSLEILCEAKKLNDA (SEQ ID NO: 118); [Sequence A]-DPSVSLEILAEAKKLNDA (SEQ ID NO: 119); [Sequence A]-DPSVSLALLAEAKKLNDA (SEQ ID NO: 120); [Sequence A]-DPSQSANLLSEAKKLNES (SEQ ID NO: 121); [Sequence A]-DPSQSTNVLGEAKKLNES (SEQ ID NO: 122); and [Sequence A]-DPSQSANVLGEAQKLNDS (SEQ ID NO: 123); ([Sequence A] is as defined herein) In certain embodiments, there is provided a VH3 binding polypeptide as disclosed herein comprising a binding module sequence C selected from the group consisting of: [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); [Sequence A]-DPSQSANLLAEAKKLNES (SEQ ID NO: 116); [Sequence A]-DPSVSLEILGEAKKLNDA (SEQ ID NO: 117); [Sequence A]-DPSVSLEILCEAKKLNDA (SEQ ID NO: 118); [Sequence A]-DPSVSLEILAEAKKLNDA (SEQ ID NO: 119); [Sequence A]-DPSVSLALLAEAKKLNDA (SEQ ID NO: 120); and [Sequence A]-DPSQSANLLSEAKKLNES (SEQ ID NO: 121); From the group consisting of: for example, [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); [Sequence A]-DPSQSANLLAEAKKLNES (SEQ ID NO: 116); [Sequence A]-DPSVSLEILGEAKKLNDA (SEQ ID NO: 117); [Sequence A]-DPSVSLEILCEAKKLNDA (SEQ ID NO: 118); [Sequence A]-DPSVSLEILAEAKKLNDA (SEQ ID NO: 119); and [Sequence A]-DPSVSLALLAEAKKLNDA (SEQ ID NO: 120); From the group consisting of: for example, [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); and [Sequence A]-DPSQSANLLAEAKKLNES (SEQ ID NO: 116); From the group consisting of: for example, [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); and [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); From the group consisting of: for example, [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); and [Sequence A] -DPSVSKAILAEAKKLNDA (SEQ ID NO: 115) The present invention provides a VH3 binding polypeptide as disclosed herein, comprising a binding module sequence C selected from the group consisting of:

[0080] In one particular embodiment, the link module sequence C comprises or consists of [sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113). In one particular embodiment, the link module sequence C comprises or consists of [sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115).

[0081] In another embodiment, vi) [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); (wherein [sequence A] is as defined herein); and vii) an amino acid sequence having at least 85% identity to the sequence defined in vi); The present invention provides a VH3 binding polypeptide comprising or consisting of a link module sequence C selected from the group consisting of:

[0082] As discussed above, polypeptides that contain minor changes compared to the above amino acid sequences without significantly affecting their tertiary structure and function also fall within the scope of the present disclosure. Thus, in some embodiments, a VH3 binding polypeptide comprising a binding module sequence C as defined above may have a sequence that is at least 87%, such as at least 89%, such as at least 91%, such as at least 93%, such as at least 95%, such as at least 97% identical to the sequence defined by vi).

[0083] In another embodiment, viii) [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); (wherein [sequence A] is as defined herein); and ix) an amino acid sequence having at least 85% identity to the sequence defined in viii); The present invention provides a VH3 binding polypeptide comprising or consisting of a link module sequence C selected from the group consisting of: In another embodiment, x) [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); (wherein [sequence A] is as defined herein), and xi) an amino acid sequence having at least 85% identity to the sequence defined in x); The present invention provides a VH3 binding polypeptide comprising or consisting of a link module sequence C selected from the group consisting of:

[0084] As discussed above, polypeptides that contain minor changes compared to the above amino acid sequences without significantly affecting their tertiary structure and function also fall within the scope of the present disclosure. Thus, in some embodiments, a VH3 binding polypeptide comprising a binding module sequence C as defined above may have a sequence that is at least 87%, such as at least 89%, such as at least 91%, such as at least 93%, such as at least 95%, such as at least 97% identical to a sequence defined by any one of viii) and x).

[0085] As described in detail in the experimental section that follows, the selection of VH3-binding polypeptide variants led to the identification of several individual amino acid sequences, said sequences also referred to herein as binding module sequence C, constituting an individual embodiment of any one of sequences vi) and viii) corresponding to the sequence at positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, for example from the group consisting of SEQ ID NOs: 1 to 40 and 40 to 82, for example from the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56, as presented in Figure 5. Thus, the sequence of an individual amino acid sequence according to sequence C corresponds to the amino acids at positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, as presented in Figure 5.

[0086] Thus, in one embodiment according to the first aspect disclosed herein, there is provided a VH3-binding polypeptide in which sequence vi) or viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, for example, from the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, for example, from the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56. In one embodiment, there is provided a VH3-binding polypeptide in which sequence viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 41, for example, from the group consisting of SEQ ID NOs: 1 to 40, for example, from the group consisting of SEQ ID NOs: 1 to 15. In one embodiment, sequence vi) or viii) is selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 43 and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51 and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54 and 56 to 82 for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 13, 15, 54 and 56. In one embodiment, there is provided a VH3-binding polypeptide in which sequence vi) or viii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82, for example, a sequence selected from the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or a sequence selected from the group consisting of SEQ ID NOs: 15, 41, 56 and 82. In one embodiment a VH3 binding polypeptide is provided, wherein sequence vi) or viii) corresponds to the sequence from position 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56.In one embodiment, a VH3-binding polypeptide is provided, in which sequence viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15. In one embodiment, a VH3-binding polypeptide is provided, in which sequence viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 15 to 41; for example, the group consisting of SEQ ID NOs: 15 to 40 or the group consisting of SEQ ID NOs: 15 to 41. In one embodiment, a VH3-binding polypeptide is provided, in which sequence viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15. In one embodiment, a VH3-binding polypeptide is provided, in which sequence vi) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NO: 42, 43 and 45 to 82; for example, the group consisting of SEQ ID NO: 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NO: 47, 48, 51 and 54 to 82; for example, the group consisting of SEQ ID NO: 47, 48, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NO: 47, 51, 54, 56 to 82; for example, the group consisting of SEQ ID NO: 54 and 56 to 82; for example, the group consisting of SEQ ID NO: 54 and 56 to 81; for example, the group consisting of SEQ ID NO: 54 and 56. In one embodiment there is provided a VH3 binding polypeptide, wherein sequence vi) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 56 to 82; for example from the group consisting of SEQ ID NOs: 56 to 81 or from the group consisting of SEQ ID NOs: 56 and 82. In one embodiment there is provided a VH3 binding polypeptide, wherein sequence vi) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NO: 55 and SEQ ID NO: 56. In one embodiment there is provided a VH3 binding polypeptide, wherein sequence viii) corresponds to a sequence from positions 8 to 54 in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.In one embodiment a VH3 binding polypeptide is provided, wherein sequence vi) corresponds to the sequence from positions 8 to 54 in SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56.

[0087] Additionally included in the disclosure are the VH3 binding polypeptides listed in Figure 5 as SEQ ID NOs: 83-87. Thus, in one embodiment, there is provided a VH3 binding polypeptide comprising a sequence selected from the group consisting of: a sequence corresponding to residues 8-54 in SEQ ID NO: 83; a sequence corresponding to residues 8-54 in SEQ ID NO: 84; a sequence corresponding to residues 8-54 in SEQ ID NO: 85; a sequence corresponding to residues 11-57 in SEQ ID NO: 86; and a sequence corresponding to residues 1-47 in SEQ ID NO: 87.

[0088] In some embodiments, the link module sequence C may form part of a polypeptide comprising an amino acid sequence further comprising sequence 1 [S1] and / or sequence 2 [S2]. In one embodiment, the link module sequence C comprises the following order from N-terminus to C-terminus: [S1]-[binding module sequence C]-[S2] There is provided a VH3 binding polypeptide as defined herein comprising sequence 1 [S1], [link module sequence C] and sequence 2 [S2], [S1] or [S2] may be present or absent, [link module sequence C] is as defined herein, and Independently of each other, [S1] is, ADNKFNK (SEQ ID NO: 124); VDAKFDK (SEQ ID NO: 125); VDNKFNK (SEQ ID NO: 126); ADAQQNKFNK (SEQ ID NO: 127); IAAKHDK (SEQ ID NO: 128); IAAQHDK (SEQ ID NO: 129); ADNNFNK (SEQ ID NO: 130); IAAKFDE (SEQ ID NO: 131); PAAKHDK (SEQ ID NO: 132); ADNAFNT (SEQ ID NO: 133); FNK; ADNRFNE (SEQ ID NO: 134); IDSKFDE (SEQ ID NO: 135); ADNRFNR (SEQ ID NO: 136); ADNKHNK (SEQ ID NO: 137); ADSKFDE (SEQ ID NO: 138); I Selected from the group consisting of DAKHDE (SEQ ID NO: 139); QQNKFNK (SEQ ID NO: 140); ADNKFHK (SEQ ID NO: 141); KFNK (SEQ ID NO: 142); ADNNFNR (SEQ ID NO: 143); AAAKHDK (SEQ ID NO: 144); IDNKFNK (SEQ ID NO: 145); IDAKFDE (SEQ ID NO: 146); DNNFNK (SEQ ID NO: 147); ADNKFNE (SEQ ID NO: 148); AAAQHDK (SEQ ID NO: 149); and AAAKFDE (SEQ ID NO: 150); [S2] is Selected from the group consisting of QAPK (sequence number 151); QAPP (sequence number 152); QAP; QAPR (sequence number 153); QAPE (sequence number 154); APK; QAPG (sequence number 155); QAIK (sequence number 156); and QA.

[0089] In one embodiment, S1 is selected from the group consisting of ADNKFNK;VDAKFDK;VDNKFNK;IAAKHDK;IAAQHDK;ADNNFNK;IAAKFDE;PAAKHDK;ADNAFNT;FNK;ADNRFNE; and IDSKFDE, for example, the group consisting of ADNKFNK;VDAKFDK;VDNKFNK;IAAKHDK;IAAQHDK;ADNNFNK;IAAKFDE; and PAAKHDK, for example, the group consisting of ADNKFNK;VDAKFDK;VDNKFNK;IAAKHDK;IAAQHDK; and ADNNFNK, for example, the group consisting of ADNKFNK;VDAKFDK;VDNKFNK;IAAKHDK; and IAAQHDK, for example, the group consisting of ADNKFNK;VDAKFDK;VDNKFNK; and IAAKHDK, for example, the group consisting of ADNKFNK;VDAKFDK;VDNKFNK; and IAAKHDK, for example, the group consisting of ADNKFNK;VDAKFDK; and VDNKFNK. In one embodiment, S1 comprises or consists of VDNKFNK (SEQ ID NO: 158). In one embodiment, S1 comprises or consists of VDAKFDK (SEQ ID NO: 160). In one embodiment, S1 comprises or consists of ADNKFNK (SEQ ID NO: 159).

[0090] In one embodiment, S2 is selected from the group consisting of QAPK; QAPP; QAP; QAPR; and QAPE, such as the group consisting of QAPK; QAPP; QAP; and QAPE, such as the group consisting of QAPK; QAPP; QAP; and QAPR, such as the group consisting of QAPK; QAPP and QAP; such as the group consisting of QAPK; and QAPP. In one embodiment, S2 comprises or consists of QAPK (SEQ ID NO: 151). In one embodiment, S2 comprises or consists of QAPP (SEQ ID NO: 152). In one embodiment, S2 comprises or consists of QAP.

[0091] In some embodiments, the VH3 binding polypeptide may be selected from a polypeptide in which sequence A forms a part of: ADNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 157); VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); ADNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 159); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 160); VDNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 161); VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162); ADNNFNK-[sequence A]-DPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 163); ADNKHNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 164); ADNKFNK-[sequence A]-DPSQSANLLAEAAPK (SEQ ID NO: 165); IAAQHDK-[sequence A]-DPSVSLEILAEAKKLNDAQAPK (SEQ ID NO: 166); ADNKFHK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 167); FNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 168); IAAQHDK-[sequence A]-DPSVSLEILCEAKKLNDAQAPK (SEQ ID NO: 169); IDAKFDE-[sequence A]-DPSVSLALLAEAKKLNDAQAPP (SEQ ID NO: 170); DNNFNK-[sequence A]-DPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 171); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAIK (SEQ ID NO: 172); ADNRFNE-[sequence A]-DPSVSKEILAEAKKLNDAQAPE (SEQ ID NO: 173); and IDAKFDE-[sequence A]-DPSVSLSLLAEAKKLNDAQAPP (SEQ ID NO: 174); ([Sequence A] is as defined herein) The amino acid sequence may be selected from the group consisting of:

[0092] In certain embodiments, the VH3 binding polypeptide comprises ADNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 157); VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); ADNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 159); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 160); VDNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 161); VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162); ADNNFNK-[sequence A]-DPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 163); ADNKHNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 164); and ADNKFNK-[sequence A]-DPSQSANLLAEAAPK (SEQ ID NO: 165); From the group consisting of; for example, ADNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 157); VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); ADNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 159); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 160); VDNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 161); and VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162); From the group consisting of; for example, ADNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 157); VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); ADNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 159); and VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 160); and VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162); for example, VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 160); VDNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 161); and VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162); From the group consisting of; for example, VDNKFNK-[Sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); and VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162) The amino acid sequence is selected from the group consisting of:

[0093] In one embodiment, the VH3 binding polypeptide comprises or consists of VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158). In one embodiment, the VH3 binding polypeptide comprises or consists of VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162).

[0094] In one embodiment, the VH3 binding polypeptide comprises the below described: xii) VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158) (wherein [sequence A] is as defined herein); xiii) an amino acid sequence having at least 86% identity to the sequence defined in xii); The amino acid sequence is selected from the group consisting of:

[0095] In one embodiment, the VH3 binding polypeptide comprises xiv) VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162) (wherein [sequence A] is as defined herein); and xv) an amino acid sequence having at least 86% identity to the sequence defined in xiv); The amino acid sequence is selected from the group consisting of:

[0096] Again, polypeptides comprising minor changes compared to the above amino acid sequences xii) or xiv) without significantly affecting their tertiary structure and function are also within the scope of the present disclosure. Thus, in some embodiments, a VH3 binding polypeptide as defined above may have a sequence which is for example at least 87%, such as at least 89%, for example at least 91%, such as at least 93%, for example at least 94%, such as at least 96%, for example at least 98% identical to the sequence defined by xii) or xiv).

[0097] Sequence xiv) in such a polypeptide may be selected from the group consisting of SEQ ID NOs: 1 to 41, for example SEQ ID NOs: 1 to 40. Sequence xii) in such a polypeptide may be selected from the group consisting of SEQ ID NOs: 42 to 82, for example SEQ ID NOs: 42 to 81. Thus, in one embodiment, sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, for example SEQ ID NOs: 1 to 40 and 42 to 81, for example SEQ ID NOs: 1 to 15 and 42 to 56. In one embodiment, sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1 to 41, for example SEQ ID NOs: 1 to 40, for example SEQ ID NOs: 1 to 15. In one embodiment, the sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54, and 56 to 81; for example, the group consisting of SEQ ID NOs: 13, 15, 54, and 56. In another embodiment, sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82; for example from the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or from the group consisting of SEQ ID NOs: 15, 41, 56 and 82. In one embodiment, sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56.In one embodiment, sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15. In one embodiment, sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 15 to 41; for example, a sequence selected from the group consisting of SEQ ID NOs: 15 to 40 or from the group consisting of SEQ ID NOs: 15 and 41. In another embodiment, sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 14 and 15. In yet another embodiment, sequence xii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 42, 43 and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56. In one embodiment, sequence xii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 56 to 82; for example, the group consisting of SEQ ID NOs: 56 to 81 or the group consisting of SEQ ID NOs: 56 and 82. In one embodiment, sequence xii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 55 and 56. In one embodiment, sequence xiv) corresponds to SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. And in another embodiment, sequence xii) corresponds to the sequence SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56.

[0098] The VH3 binding polypeptide may also be selected from a sequence according to any one of SEQ ID NOs: 83 to 87. Thus, in one embodiment, said VH3 binding polypeptide disclosed herein comprises a sequence selected from the group consisting of: a sequence corresponding to residues 1 to 58 in SEQ ID NO: 83; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 84; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 85; a sequence corresponding to residues 3 to 60 in SEQ ID NO: 86 and a sequence corresponding to residues 1 to 51 in SEQ ID NO: 87, or a sequence selected from the group consisting of: a sequence corresponding to residues 1 to 58 in SEQ ID NO: 83; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 84; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 85; a sequence corresponding to residues 1 to 63 in SEQ ID NO: 86 and a sequence corresponding to residues 1 to 51 in SEQ ID NO: 87.

[0099] Further polypeptide domains having the same functionality may be attached to a VH3 binding polypeptide as defined herein, for example any VH3 binding polypeptide as defined in any one of i) to xv), thus achieving a VH3 binding polypeptide multimer. Such multimers may be useful, in particular, to increase the efficiency of binding VH3 regions, for example to ensure that more VH3 region-containing polypeptides are bound to the multimer, as compared to a monomeric variant of the VH3 binding polypeptide (in other words, the VH3 binding polypeptide). Thus, in a second aspect of the present disclosure, a VH3 binding polypeptide multimer is provided. Said multimer is understood to comprise at least two VH3 binding polypeptides as disclosed herein as monomeric units, the amino acid sequences of which may be the same or different. Thus, in one embodiment, a VH3 binding polypeptide multimer is provided, in which each monomer of the multimer comprises a VH3 binding polypeptide independently selected from any VH3 binding polypeptide as defined herein. In another embodiment, there is provided a VH3 binding polypeptide multimer, wherein each monomer of the multimer comprises a sequence A independently selected from any sequence A defined herein.

[0100] A multimeric form of a polypeptide may comprise a suitable number of domains, each having VH3 binding activity, each forming a monomer within the multimer. In one embodiment, the multimer is selected from the group consisting of a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer, e.g., selected from the group consisting of a dimer, tetramer, pentamer, hexamer, heptamer and octamer, e.g., selected from the group consisting of a dimer, tetramer and hexamer. In one particular embodiment, the multimer is a hexamer. In one particular embodiment, the multimer is a tetramer. In one embodiment, the multimer is a linear multimer comprising monomers arranged sequentially. In one embodiment, the multimer is a linear polypeptide comprising monomers arranged sequentially. In one embodiment, each monomer is arranged such that its C-terminus is adjacent to the N-terminus of the monomer immediately following it. In one embodiment, at least one monomer is positioned such that its C-terminus is adjacent to the C-terminus of the monomer immediately following that monomer.

[0101] The monomers in a multimer may have the same amino acid sequence, or may have different amino acid sequences. Thus, the monomers may have any amino acid sequence of a VH3 binding polypeptide as defined herein, for example, all monomers in a multimer may have different sequences, or only a subset of monomers in a multimer may have different amino acid sequences, the others having the same sequence. In other words, the VH3 binding polypeptides of the invention may form homo- or heteromultimers, e.g., homodimers or heterodimers. In one embodiment, a homomeric VH3 binding polypeptide multimer is provided. In another embodiment, a heteromeric VH3 binding polypeptide multimer is provided. The heteromer may comprise at least two or at least three different sequences A, each sequence A being as defined herein. In one embodiment, the multimer is a heteromer comprising at least two or at least three different VH3 binding polypeptides, each as defined herein. In one embodiment, the multimer is a homomer comprising four VH3 binding polypeptides as defined herein. In another embodiment, the multimer is a homomer comprising six VH3 binding polypeptides as defined herein. In a particular embodiment, the multimer is a homomer or heteromer comprising at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight monomers selected from the group consisting of SEQ ID NOs: 1 to 82, such as from the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, such as from the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56; such as from the group consisting of SEQ ID NOs: 1 to 41, such as from the group consisting of SEQ ID NOs: 1 to 40, such as from the group consisting of SEQ ID NOs: 1 to 15.In a particular embodiment, the multimer is selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54, and 56 to 82; , the group consisting of SEQ ID NOs: 13, 15 to 40, 54 and 56 to 81; for example, selected from the group consisting of SEQ ID NOs: 13, 15, 54 and 56, or the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82; for example, selected from the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or from the group consisting of SEQ ID NOs: 15, 41, 56 and 82; or selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56. In a particular embodiment, the multimer is a homomer or heteromer comprising at least 3, such as at least 4, for example at least 5, such as at least 6, for example at least 7, for example at least 8 monomers selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example the group consisting of SEQ ID NOs: 13, 15 to 41; for example the group consisting of SEQ ID NOs: 13, 15 to 40; for example the group consisting of SEQ ID NOs: 13 and 15; or the group consisting of SEQ ID NOs: 15 to 41; for example selected from the group consisting of SEQ ID NOs: 15 to 40 or from the group consisting of SEQ ID NOs: 15 and 41; or selected from the group consisting of SEQ ID NOs: 14 and 15.In one embodiment, the multimer is a homomer or heteromer comprising at least three, for example at least four, for example at least five, for example at least six, for example at least seven, for example at least eight monomers selected from the group consisting of SEQ ID NOs: 42, 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54, 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56; or the group consisting of SEQ ID NOs: 56 to 82; for example, selected from the group consisting of SEQ ID NOs: 56 to 81 or from the group consisting of SEQ ID NOs: 56 and 82; or the group consisting of SEQ ID NOs: 55 and 56. In one embodiment, the multimer is a homomer or heteromer comprising at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight monomers selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15; or selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. In one embodiment, the multimer comprises the sequence of SEQ ID NO: 13. In one embodiment, the multimer comprises the sequence of SEQ ID NO: 14. In one embodiment, the multimer comprises the sequence of SEQ ID NO: 15. In the attached examples, multimers of each of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are described. The multimers exhibit desirable properties in terms of VH3 affinity and stability when exposed to alkaline washes. Furthermore, the multimers have low or no affinity for the Fc region of trastuzumab. Specific examples of such multimers are according to SEQ ID NO: 175, 176 and 177. SEQ ID NO: 175 comprises four copies of SEQ ID NO: 13 and is therefore a tetramer thereof. SEQ ID NO: 175 contains four instances of SEQ ID NO: 15 and is therefore a tetramer thereof. SEQ ID NO: 177 contains four instances of SEQ ID NO: 15 and is therefore a hexamer thereof.

[0102] It is understood that the multimer may comprise any one of SEQ ID NOs: 83 to 87. In one embodiment, the multimer is a homomer or heteromer comprising at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight monomers selected from the group consisting of: a sequence corresponding to residues 1 to 58 in SEQ ID NO: 83; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 84; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 85; a sequence corresponding to residues 3 to 60 in SEQ ID NO: 86 and a sequence corresponding to residues 1 to 51 in SEQ ID NO: 87, or selected from the group consisting of: a sequence corresponding to residues 1 to 58 in SEQ ID NO: 83; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 84; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 85; a sequence corresponding to residues 1 to 63 in SEQ ID NO: 86 and a sequence corresponding to residues 1 to 51 in SEQ ID NO: 87.

[0103] It is understood that the monomers in the multimers discussed herein, comprising sequence A or comprising a VH3 binding polypeptide as disclosed herein, can be directly coupled to each other or spaced apart by a linker sequence. The skilled artisan will understand that the presence or absence of a linker sequence can vary between different monomer moieties in a multimer, and that the sequence of each individual linker, if a linker is present, can be the same or different. Thus, in one embodiment, the VH3 binding polypeptide multimer comprises at least one linker. As the skilled artisan will understand, the construction of multimers, for example as fusion proteins, frequently involves the use of linkers between the monomer moieties to be fused. The skilled artisan will be aware of different types of linkers, for example flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers, with different properties. Linkers can be used, for example, to increase the stability or improve the folding of the fusion protein, to increase expression, or to improve activity, affinity and / or binding capacity. Thus, in one embodiment, the VH3 binding polypeptide multimer as defined herein further comprises at least one linker. In one embodiment, a linker is present between each monomer in the multimer. The linker may, for example, be selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. Alternatively, the linker may be a non-peptidic linker. Thus, the VH3 binding polypeptides disclosed herein may be directly linked to each other by a peptide bond between the C-terminus and the N-terminus of the polypeptide. Alternatively, two or more monomers in the multimer, in other words monomeric units or moieties, may be linked by an element comprising an oligomeric or polymeric species, for example an element comprising 15 or up to 30 amino acids, for example 1-5, 1-10, or 5-10 amino acids. In one embodiment, said linker comprises up to 15 amino acid residues. The nature of such a link preferably does not destabilize the spatial conformation of the protein unit, i.e. of the VH3 binding polypeptide monomers in the multimer. This can be achieved, for example, by avoiding the presence of prolines in the linker.Moreover, said linker is preferably also sufficiently stable in an alkaline environment so as not to impair the properties of the protein unit. For this purpose, it is advantageous that the linker does not contain asparagine. It may be further advantageous that the linker does not contain glutamine. The multimer may further comprise a number of amino acid residues at the N-terminus, which may be derived from the cloning step or may constitute residues from a cleaved signal sequence. The number of further amino acid residues may be, for example, 15 or less, for example 10 or less, or 5 or less.

[0104] Those skilled in the art will appreciate that various modifications and / or additions may be made to a VH3 binding polypeptide or VH3 binding polypeptide multimer according to any embodiment disclosed herein to adapt the polypeptide or multimer for a particular application without departing from the scope of the disclosure.

[0105] For example, in one embodiment, a VH3 binding polypeptide or VH3 binding polypeptide multimer as described herein is provided which is extended by and / or comprises further amino acids at the C-terminus and / or N-terminus. Such a polypeptide or multimer is understood as a polypeptide or multimer having one or more further amino acid residues at the beginning and / or end of the polypeptide chain, i.e. at the N-terminus and / or C-terminus of the polypeptide or multimer. For example, said further amino acid residues may be sequence A according to i), ii) or iii), binding module sequence C according to vi), vii), viii), ix), x), xi), or sequence xii), xiii), xiv), or xv) at the N-terminus and / or C-terminus. Thus, a VH3 binding polypeptide as defined herein may comprise any suitable number of further amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more further amino acid residues. The amino acid residues may individually or collectively improve the in vitro production, purification, stabilization, or coupling of the polypeptide to a substrate of interest, e.g., a solid support, e.g., a solid support as described in connection with the embodiment of a separation matrix.

[0106] The further amino acid residues can be coupled to the VH3 binding polypeptide or multimer by chemical conjugation (using known organic chemistry methods) or by any other means, e.g. by expression of the VH3 binding polypeptide or multimer as a fusion protein, or can be linked in any other way, either directly or via a linker, e.g. an amino acid linker as described above.

[0107] In some embodiments, the VH3 binding polypeptide and / or multimer further comprises one or more coupling elements at the C-terminus or N-terminus selected from the group consisting of a cysteine ​​residue, a number of lysine residues and a number of histidine residues, as disclosed above. The coupling element may for example be a single cysteine ​​at the C-terminus. The coupling element may be directly linked to the C-terminus or N-terminus or may be linked via a linker comprising up to 15 amino acids, for example 1-5, 1-10 or 5-10 amino acids. This stretch is also preferably stable enough in an alkaline environment so as not to impair the properties of the protein. To that end, it is advantageous if the stretch does not contain asparagine. It may be even more advantageous if the stretch does not contain glutamine. An advantage of having a C-terminal cysteine ​​is that end-point coupling of the protein can be achieved by reaction of the cysteine ​​thiol with an electrophilic group on the support. This provides excellent mobility of the coupled protein, which is important for binding capacity.

[0108] Those skilled in the art will recognize that there are many ways to produce polypeptides.Thus, in one embodiment, there is provided a VH3 binding polypeptide multimer as disclosed herein, wherein said VH3 binding polypeptide monomer is expressed as a fusion protein.In another embodiment, there is provided a VH3 binding polypeptide multimer as disclosed herein, wherein said VH3 binding polypeptide monomer units are covalently linked to each other.

[0109] As used in this disclosure, the terms "VH3 binding" and "binding affinity for VH3" refer to a property of a polypeptide that can be tested, for example, by ELISA, or by using surface plasmon resonance (SPR) technology.

[0110] VH3 binding affinity can be tested in an experiment in which a VH3-containing polypeptide or a fragment thereof (see also samples below) is immobilized on a sensor chip of a surface plasmon resonance (SPR) instrument and a sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument and a sample containing VH3 or a fragment thereof is passed over the chip. The skilled person can then interpret the results obtained by such an experiment to establish at least a qualitative measure of the binding affinity of the polypeptide for VH3. For example, the K D Surface plasmon resonance may also be used to determine binding values, if a quantitative measurement is desired. Binding values ​​may be determined, for example, in a Biacore™ (Cytiva™) or ProteOn™ XPR 36 (Bio-Rad™) instrument. A VH3-containing polypeptide or a fragment thereof is suitably immobilized on the sensor chip of the instrument, and a sample of the polypeptide whose affinity is to be determined is prepared by serial dilution and randomly injected. A VH3-containing Fab may be used as a target for the assessment of VH3 binding affinity, with K D Values ​​can be calculated from the results, for example using the 1:1 Langmuir binding model of the BIAevaluation 4.1 software, or other suitable software provided by the instrument manufacturer. Those skilled in the art will appreciate that in the above experimental setup, the VH3-containing Fab can be passed over the chip or immobilized on the sensor chip.

[0111] As explained above, the VH3 binding polypeptides and VH3 binding polypeptide multimers of the present disclosure can bind to the VH3 region of trastuzumab. Thus, in one embodiment, the K D The value is up to 1×10 -6 M, for example, up to 1×10 -7 M, for example, up to 1×10-8 M, for example, up to 1×10 -9 M, for example, up to 1×10 -10 M, for example, up to 1×10 -11 There is provided a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein that is capable of binding to VH3 such that K D The value may be calculated based on the interaction of a VH3 binding polypeptide or VH3 binding polypeptide multimer disclosed herein with a VH3-containing Fab.

[0112] The VH3 binding polypeptide and / or VH3 binding polypeptide multimer may have a K similar to SEQ ID NO: 88 and / or SEQ ID NO: 89 for interaction with VH3. D In particular, the K D The values ​​are the K D For example, the K of the interaction of SEQ ID NO:88 and / or SEQ ID NO:89 with a particular VH3 may differ by an order of magnitude. D The value is 9 × 10 in an assay. -10 M, the K of the interaction of the VH3-binding polypeptide or VH3-binding polypeptide multimer with VH3 D The value was 9 × 10 in that assay. -9 M~9×10 -11 The K obtained using the same assay can be in the range of 100 M. D It will be appreciated that it may be useful to compare values, and that while some variation may occur between different assays, intra-assay comparisons will generally demonstrate the same trends regardless of the assay utilized. The assays may differ, for example, depending on whether it is a VH3-containing polypeptide or a VH3-binding polypeptide that is immobilized on the chip. In one embodiment, the K D In a different embodiment, the K value for interaction with VH3 is measured in an assay in which the VH3 binding polypeptide is immobilized on a chip.D The value is measured in an assay in which the VH3-containing polypeptide is immobilized on a chip. In one embodiment, the VH3-containing polypeptide is a VH3-containing Fab.

[0113] In another embodiment, the K D The value is the K D There is provided a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein capable of binding to VH3 such that the K D The value is the K D In one embodiment said VH3 binding polypeptide or VH3 binding polypeptide multimer is capable of binding to VH3 such that the K D The values ​​are the K D The K of interaction with VH3 is at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%. D Values ​​can be tested, for example, using Fab-containing VH3 or using full-length antibodies. For example, measurements can be performed using the conditions described in Examples 1, 2 or 3. Those skilled in the art will appreciate that other VH3-containing polypeptides can be used, where appropriate.

[0114] Similarly, the terms "Fc binding" and "binding affinity to Fc" as used in this disclosure refer to a property of a polypeptide that can be tested, for example, by ELISA or by using surface plasmon resonance (SPR) technology. Fc binding affinity can be tested in an experiment in which Fc or a fragment thereof is immobilized on the sensor chip of a surface plasmon resonance (SPR) instrument and a sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested (in this case, a VH3 binding polypeptide) can be immobilized on the sensor chip of the instrument and a sample containing Fc or a fragment thereof is passed over the chip. Those skilled in the art will appreciate that if binding to Fc is lost or significantly reduced, essentially an absence of binding should be detected. Those skilled in the art can then interpret the results obtained by such an experiment to establish at least a qualitative measure of the binding affinity of the polypeptide to Fc. For example, the K D If a quantitative measurement is desired to determine values, surface plasmon resonance may also be used in a manner similar to that described above for VH3 binding.

[0115] As explained in connection with the first aspect of the disclosure, the VH3-binding polypeptides and VH3-binding polypeptide multimers defined herein may have significantly reduced or eliminated binding affinity to the Fc of trastuzumab. The skilled artisan will appreciate that the VH3-binding polypeptides and VH3-binding polypeptide multimers do not significantly interact with the Fc and therefore have low binding capacity to the Fc.

[0116] In one embodiment, Fc is 1×10 -4 Less than M, e.g. 1×10 -3 K for interactions less than M D In another aspect, there is provided a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein which is unable to bind at the same value.

[0117] Furthermore, the VH3 binding polypeptides and multimers thereof disclosed herein exhibit improved alkaline wash stability, for example in embodiments using said VH3c binding polypeptides or multimers for the separation or isolation of antibodies / immunoglobulins having VH3 regions, high alkaline stability allows for the use of highly alkaline conditions during washing, which is essential for long term repeated use in bioprocess separation settings.

[0118] In one embodiment there is provided a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein which retains at least 19%, such as at least 20%, such as at least 21%, such as at least 23%, such as at least 25%, such as at least 27%, such as at least 29%, such as at least 31%, such as at least 33%, such as at least 35%, such as at least 37%, such as at least 39%, such as at least 41%, such as at least 43%, such as at least 45%, such as at least 47%, such as at least 48%, such as at least 50% of its original VH3 binding capacity after at least one washing step with 0.5 M NaOH. In one embodiment said washing step comprises exposure to, such as incubation with, 0.5 M NaOH for approximately 2 to 15 minutes, such as 5 to 15 minutes, such as for approximately 5 minutes or for approximately 10 minutes or for approximately 15 minutes. For example, the incubation time with 0.5 M NaOH can be 5±0.5 min, 6±0.5 min, 7±0.5 min, or 8±0.5 min, 9±0.5 min, 10±0.5 min, 11±0.5 min, 13±0.5 min, 14±0.5 min, 14±0.5 min or 15±0.5 min. The incubation time can be longer, such as for example about 20 min or about 30 min or even 60 min or more. The skilled person is familiar with the appropriate time frame for washing an affinity column with NaOH. In one embodiment the VH3 binding polypeptide or VH3 binding polypeptide multimer disclosed herein retains at least 19%, such as at least 20%, such as at least 21%, such as at least 23%, such as at least 25%, such as at least 27%, such as at least 29%, such as at least 31%, such as at least 33%, such as at least 35%, such as at least 37%, such as at least 39%, such as at least 41%, such as at least 43%, such as at least 45%, such as at least 47%, such as at least 48%, such as at least 50% of its original VH3 binding capacity after at least 10, 20, 40, 50, 75 or 100 washing steps with 0.5 M NaOH.For example, each washing step may be for 10 minutes, and therefore the exposure time (and therefore incubation time) of a VH3 binding polypeptide or VH3 binding polypeptide multimer disclosed herein to NaOH will be 1000 minutes if 100 washing steps are performed.

[0119] In one embodiment, a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein is provided which retains at least 19%, such as at least 20%, such as at least 21%, such as at least 23%, such as at least 25%, such as at least 27%, such as at least 29%, such as at least 31%, such as at least 33%, such as at least 35%, such as at least 37%, such as at least 39%, such as at least 41%, such as at least 43%, such as at least 45%, such as at least 47%, such as at least 48%, such as at least 50% of its original VH3 binding capacity after 100 repeated binding cycles followed by washing with 0.5 M NaOH. It is understood that each binding cycle is followed by a washing step. The washing step may be, for example, a contact with 0.5 M NaOH, for example, incubation for approximately 5 to 15 minutes, such as approximately 5 minutes or 10 minutes or 15 minutes. For example, the time of incubation in contact with 0.5 M NaOH can be 5±0.5 minutes, 6±0.5 minutes, 7±0.5 minutes, or 8±0.5 minutes, 9±0.5 minutes, 10±0.5 minutes, 11±0.5 minutes, 13±0.5 minutes, 14±0.5 minutes, 14±0.5 minutes or 15±0.5 minutes.

[0120] In one embodiment there is provided a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein which exhibits at least 35%, such as at least 40%, for example at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95% of the alkaline stability of SEQ ID NO: 88 or SEQ ID NO: 89 after incubation in 0.5 M NaOH, for example after repeated incubation with 0.5 M NaOH. In one embodiment said VH3 binding polypeptide or VH3 binding polypeptide multimer has an alkaline stability after incubation in 0.5 M NaOH that is at least 60%, such as at least 70%, such as at least 80%, for example at least 85%, such as at least 89%, for example at least 90%, such as at least 95%, for example at least 98%, such as at least 99% of the alkaline stability of SEQ ID NO: 88. In one embodiment said VH3 binding polypeptide or VH3 binding polypeptide multimer has an alkaline stability after incubation in 0.5 M NaOH that is at least 60%, such as at least 70%, such as at least 80%, for example at least 85%, such as at least 89%, for example at least 90%, for example at least 95%, such as at least 98%, for example at least 99% of the alkaline stability of SEQ ID NO: 89. In one embodiment said VH3 binding polypeptide or VH3 binding polypeptide multimer has an alkaline stability after incubation in 0.5 M NaOH that is at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 89%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% of the alkaline stability of SEQ ID NO: 88 and SEQ ID NO: 89. The skilled person will appreciate that the alkaline wash stability of the polypeptides disclosed herein may be tested and compared against the alkaline wash stability of SEQ ID NO: 88 or 89, for example as described in the accompanying examples.

[0121] In one embodiment there is provided a VH3 binding polypeptide or VH3 binding polypeptide multimer as disclosed herein which retains at least 19%, such as at least 20%, such as at least 21%, such as at least 23%, such as at least 25%, such as at least 27%, such as at least 29%, such as at least 31%, such as at least 33%, such as at least 35%, such as at least 37%, such as at least 39%, such as at least 41%, such as at least 43%, such as at least 45%, such as at least 47%, such as at least 48%, such as at least 50% of its original VH3 binding capacity after incubation in 0.5M NaOH. In one embodiment said incubation is for at least 12 hours, such as at least 24 hours, such as at least 36 hours, such as at least 48 hours, such as at least 72 hours, such as at least 96 hours or more. Said incubation may for example be at 22+ / -2°C. Thus, the extent of VH3 binding capacity may, for example, be assessed following incubation of the VH3 binding polypeptide or multimer thereof in 0.5 M NaOH at 22° C. for 24 hours.

[0122] In a third aspect of the disclosure, there is provided a polynucleotide encoding a VH3 binding polypeptide or VH3 binding polypeptide multimer as described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector.

[0123] Also included in the disclosure is a method for producing a VH3 binding polypeptide or VH3 binding polypeptide multimer described herein, comprising culturing the host cell under conditions permissive for expression of the polypeptide or multimer from its expression vector, and isolating the polypeptide or multimer.

[0124] Alternatively, the VH3 binding polypeptides or multimers disclosed herein may be produced by abiotic peptide synthesis methods using amino acids and / or amino acid derivatives with protected reactive side chains, which abiotic peptide synthesis methods include: - stepwise coupling of amino acids and / or amino acid derivatives to form a polypeptide according to the first aspect or a multimer according to the second aspect, said polypeptide having protected reactive side chains, - removal of protecting groups from reactive side chains of the polypeptide or multimer, and - Folding of polypeptides or multimers in aqueous solution Includes.

[0125] In another aspect, the present invention discloses a separation matrix comprising a VH3 binding polypeptide according to any of the embodiments disclosed above coupled to a solid support. Such a matrix is ​​useful, for example, for the separation of immunoglobulins or other VH3-containing proteins from a sample. The VH3 binding polypeptides or multimers thereof disclosed herein have binding affinity for VH3 but low or no binding affinity for Fc, and the separation matrix may allow efficient separation of VH3-containing proteins from any Fc-containing proteins or impurities. As mentioned above, the VH3 binding polypeptides exhibit improved alkaline stability, allowing the use of highly alkaline conditions during washing of the separation matrix, which is essential for long-term repeated use in bioprocess separation settings. The alkaline stability of the matrix may be evaluated by measuring the immunoglobulin binding capacity or VH3 binding capacity before and after incubation in an alkaline solution at a specified temperature, e.g., 22+ / -2°C. Incubations may be carried out, for example, in 0.5 M or 1.0 M NaOH for several 5, 10, or 15 minute cycles, for example 50, 100, 200 or 300 cycles, corresponding to total incubation times of, for example, 12.5, 25, 50 or 75 h.

[0126] The VH3 binding capacity of the matrix after 96-100 cycles of 15 minute incubation in 0.5 M NaOH at 22+ / -2°C or a total incubation time of 24 or 25 h may be at least 80%, such as at least 85%, at least 90%, or at least 95% of the VH3 capacity before incubation. The capacity of the matrix after a total incubation time of 24 h in 1.0 M NaOH at 22+ / -2°C may be at least 70%, such as at least 80%, or at least 90% of the VH3 capacity before incubation. The VH3 binding capacity of the separation matrix may be measured as the dynamic binding capacity (DBC). Thus, the dynamic binding capacity of a separation matrix after 96-100 cycles of 15 minute incubation in 0.5 M NaOH at 22+ / -2°C or a total incubation time of 24 or 25 h may be at least 80%, such as at least 85%, at least 90%, or at least 95% of its DBC before incubation. The DBC of a separation matrix after a total incubation time of 24 h in 1.0 M NaOH at 22+ / -2°C may be at least 70%, such as at least 80%, or at least 90% of the DBC before incubation.

[0127] In one embodiment, the separation matrix comprises a VH3 binding polypeptide multimer, each monomer of which comprises a VH3 binding polypeptide as defined herein. The multimer, which is a homomultimer or a heteromultimer, may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer. For example, the separation matrix may comprise a hexamer of a VH3 binding polypeptide coupled to a solid support. For example, the separation matrix may comprise a tetramer of a VH3 binding polypeptide coupled to a solid support. Each monomer of the VH3 binding polypeptide multimer may comprise a sequence A independently selected from any sequence A as defined herein. In particular, the separation matrix may comprise a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, for example from the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, for example from the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56; for example from the group consisting of SEQ ID NOs: 1 to 41, for example from the group consisting of SEQ ID NOs: 1 to 40, for example from SEQ ID NOs: 1 to 15. In one embodiment, the separation matrix is ​​selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54, and 56 to 82 for example, a sequence selected from the group consisting of SEQ ID NOs: 13, 15 to 41, 54, and 56 to 82; for example, a sequence selected from the group consisting of SEQ ID NOs: 13, 15 to 40, 54, and 56 to 81; for example, a sequence selected from the group consisting of SEQ ID NOs: 13, 15, 54, and 56; or a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82; for example, a sequence selected from the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or from the group consisting of SEQ ID NOs: 15, 41, 56, and 82; or a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55, and 56.In one embodiment, the separation matrix comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15; or the group consisting of SEQ ID NOs: 15 to 41; for example, selected from the group consisting of SEQ ID NOs: 15 to 40 or from the group consisting of SEQ ID NOs: 15 and 41; or selected from the group consisting of SEQ ID NOs: 14 and 15. In one embodiment, the separation matrix comprises a sequence selected from the group consisting of SEQ ID NOs: 42, 43 and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; or the group consisting of SEQ ID NOs: 56 to 82; for example, the group consisting of SEQ ID NOs: 56 to 81 or the group consisting of SEQ ID NOs: 56 and 82; or the group consisting of SEQ ID NOs: 55 and 56. In one embodiment, the separation matrix comprises a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15; or a sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. In one embodiment, the separation matrix comprises the sequence of SEQ ID NO: 13. In one embodiment, the separation matrix comprises the sequence of SEQ ID NO: 14. In one embodiment, the separation matrix comprises a VH3 binding polypeptide multimer, optionally a hexamer or tetramer, wherein each monomer of the multimer comprises a sequence corresponding to SEQ ID NO: 15.

[0128] The separation matrix may comprise a sequence selected from the group consisting of SEQ ID NOs: 83-87. In one embodiment, the separation matrix comprises a VH3 binding polypeptide multimer, optionally a hexamer or tetramer, each monomer of the multimer comprising a sequence corresponding to any one of the aforementioned sequences. In one embodiment, the separation matrix comprises a VH3 binding polypeptide multimer, optionally a hexamer or tetramer, each monomer of the multimer comprising a sequence corresponding to SEQ ID NO: 13. In one embodiment, the separation matrix comprises a VH3 binding polypeptide multimer, optionally a hexamer or tetramer, each monomer of the multimer comprising a sequence corresponding to SEQ ID NO: 14. In one embodiment, the separation matrix comprises a VH3 binding polypeptide multimer, optionally a hexamer or tetramer, each monomer of the multimer comprising a sequence corresponding to SEQ ID NO: 15. The separation matrix may comprise a sequence selected from the group consisting of SEQ ID NOs: 175-177, for example from the group consisting of SEQ ID NOs: 176 and 177. In one embodiment, the separation matrix comprises SEQ ID NO: 175. In one embodiment, the separation matrix comprises SEQ ID NO: 176. In one embodiment, the separation matrix comprises SEQ ID NO: 177. As the skilled artisan will appreciate, the expressed VH3 binding polypeptide, in monomeric or multimeric form, should be purified to an appropriate extent before immobilization to a support. Such purification methods are well known in the art, and immobilization of protein-based ligands to a support is easily performed using standard methods. Suitable methods and supports are discussed in more detail below and are disclosed, for example, in WO 16079033, which is incorporated herein by reference.

[0129] The solid support of the matrix according to the invention can be of any known suitable type. As a non-limiting example, conventional affinity separation matrices are based on polymers that are frequently organic and expose a hydrophilic surface to the aqueous medium used, i.e. hydroxyl (-OH), carboxyl (-COOH), carboxamide (-CONH2, possibly N-substituted), amino (-NH2, possibly substituted), oligo or polyethyleneoxy groups on their outer and, if present, on the inner surface. The solid support can suitably be porous. Porosity can be expressed as a Kav or Kd value (fraction of pore volume available for a probe molecule of a certain size) measured by inverse size exclusion chromatography, for example according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp. 6-13 (Non-Patent Document 2). By definition, both Kd and Kav values ​​are always in the range of 0 to 1. The Kav value, measured using dextran of Mw 110 kDa as probe molecule, may advantageously be between 0.6 and 0.95, for example between 0.7 and 0.90 or between 0.6 and 0.8. The advantage is that the support has a large proportion of pores that can accommodate both the polypeptide / multimer of the invention and the immunoglobulins that bind to the polypeptide / multimer, while still providing mass transport of the immunoglobulins to and from the binding site.

[0130] The polypeptide or multimer can be attached to the support via conventional coupling techniques, for example, using thiol, amino and / or carboxy groups present in the ligand. Bisepoxide, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS), etc. are well-known coupling reagents. Between the support and the polypeptide / multimer, a molecule known as a spacer may be introduced, which improves the availability of the polypeptide / multimer and facilitates the chemical coupling of the polypeptide / multimer to the support. Depending on the nature of the polypeptide / multimer and the coupling conditions, the coupling can be a multipoint coupling (e.g., via multiple lysines) or a single point coupling (e.g., via a single cysteine).

[0131] Alternatively, the polypeptides / multimers can be attached to the support by non-covalent attachment, for example by physical adsorption or biospecific adsorption.

[0132] In some embodiments, the matrix comprises 5-25, e.g. 5-20 mg / ml, 5-15 mg / ml, 5-11 mg / ml or 6-11 mg / ml of polypeptide or multimer coupled to the support. The amount of coupled polypeptide / multimer can be controlled by the concentration of polypeptide / multimer used in the coupling process, by the activation and coupling conditions used and / or by the pore structure of the support used. As a general rule, the absolute binding capacity of the matrix increases with the amount of coupled polypeptide / multimer, at least up to the point where the pores are significantly constricted by the coupled polypeptide / multimer. The relative binding capacity per mg of coupled polypeptide / multimer decreases at higher coupling levels, resulting in a cost-benefit optimum within the ranges specified above.

[0133] In certain embodiments, the polypeptide or multimer is coupled to the support via a thioether bond. Methods for performing such coupling are well known in the art and can be easily performed by those skilled in the art using standard techniques and equipment. Thioether bonds are flexible and stable, and are generally suitable for use in affinity chromatography. In particular, when the thioether bond is via a terminal or near-terminal cysteine ​​residue on the polypeptide or multimer, the mobility of the coupled polypeptide / multimer is increased, resulting in improved binding capacity and binding kinetics. In some embodiments, the polypeptide / multimer is coupled via a C-terminal cysteine ​​provided on the protein. It allows efficient coupling of cysteine ​​thiols to electrophilic groups on the support, such as epoxide groups, halohydrin groups, etc., resulting in thioether cross-link coupling.

[0134] In certain embodiments, the solid support comprises a polyhydroxypolymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc. Polysaccharides are hydrophilic in nature, have a low degree of non-specific interactions, provide a high content of reactive (activatable) hydroxyl groups, and are generally stable to alkaline washing solutions used in bioprocessing. In some embodiments, the support comprises agar or agarose. The supports used in the present invention can be easily prepared according to standard methods, for example, inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964) (Non-Patent Document 3)). Alternatively, the base matrix is ​​a commercially available product, for example, cross-linked agarose beads sold under the name SEPHAROSE™ FF (Cytiva™). In one embodiment that is particularly advantageous for large-scale separations, the support is adapted to increase its stiffness, thus making the matrix more suitable for high flow rates, using the methods described in U.S. Pat. No. 6,602,990 or U.S. Pat. No. 7,396,467, the entireties of which are incorporated herein by reference.

[0135] In certain embodiments, the support, for example, polysaccharide or agarose support, is crosslinked, for example, by hydroxyalkyl ether crosslinking. The crosslinker reagent that creates such crosslinking can be, for example, epihalohydrin, such as epichlorohydrin, diepoxide, such as butanediol diglycidyl ether, allylation reagent, such as allyl halide or allyl glycidyl ether. Crosslinking is beneficial to the rigidity of the support and improves its chemical stability. Hydroxyalkyl ether crosslinking is alkaline stable and does not cause significant nonspecific adsorption.

[0136] Alternatively, the solid support is based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkylacrylate, polyhydroxyalkylmethacrylate, polyacrylamide, polymethacrylamide, etc. In the case of hydrophobic polymers, such as divinyl and monovinyl substituted benzene based matrices, the surface of the matrix is ​​frequently hydrophilized in order to expose the hydrophilic groups defined above to the surrounding aqueous liquid. Such polymers are easily produced according to standard methods, see for example "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988) (Non-Patent Document 4)). Alternatively, commercially available products, such as SOURCE™ (Cytiva™), can be used. Alternatively, the solid support according to the invention comprises an inorganic support, such as silica, zirconium oxide, etc.

[0137] In one embodiment, the solid support has the form of a porous monolith. In an alternative embodiment, the support is in the form of beads or particles, which may be porous or non-porous. Solid supports in the form of beads or particles can be used as packed beds or in suspension form. Suspension forms include what are known as expanded beds and pure suspensions, in which the particles or beads are free to move. In the case of monoliths, packed beds and expanded beds, the separation procedure generally follows conventional chromatography with a concentration gradient. In the case of pure suspensions, a batchwise mode is used.

[0138] In one embodiment, the separation matrix is ​​a convection-based chromatography matrix. Such a convection-based chromatography matrix may comprise a porous polymer membrane, a fibrous support or a fibrous substrate. Examples of porous polymer membranes include Mustang™ membranes from Pall and Sarutobind™ membranes from Sartorius. The fibrous support may be electrospun polymeric or cellulose fibers, optionally nonwoven fibers. Thus, the fibrous support may be a fibrous nonwoven polymer matrix. The fibers contained in the fibrous support have a cross-sectional diameter of 10-1000 nm, for example, 200-800 nm, 200-400 nm or 300-400 nm. Such a fibrous support may be found in HiTrap Fibro™ units (Cytiva™). Alternative fibrous supports are disclosed, for example, in WO 2019 / 137869 A1 and WO 2018 / 011600 A1.

[0139] In yet other embodiments, the solid support is in another form, such as a surface, a chip, a capillary, or a filter (eg, a membrane or depth filter matrix).

[0140] In another aspect, the present invention provides a method for isolating immunoglobulins or fragments thereof using a separation matrix as disclosed above. In some embodiments, the method comprises the steps of: a) contacting a liquid sample containing immunoglobulins or fragments thereof with a separation matrix as disclosed above. The method may further comprise the steps of b) washing said separation matrix with a washing solution, c) eluting the immunoglobulins from the separation matrix with an elution solution, and optionally d) washing the separation matrix with a washing solution. Alternatively, the washing solution may be referred to as a cleaning in place (CIP) solution. The contact (incubation) time may be at least 10 minutes.

[0141] The method may comprise, prior to step a), providing an affinity separation matrix according to any of the previous embodiments and providing a solution comprising an immunoglobulin or a fragment thereof and at least one other substance as a liquid sample, and, after step c), recovering the eluate and optionally subjecting the eluate to a further separation step, for example by anion or cation exchange chromatography, multimodal chromatography and / or hydrophobic interaction chromatography. Suitable compositions of liquid samples, washing solutions and eluates, as well as general conditions for carrying out the separation, are well known in the art of affinity chromatography, and in particular in the art of Protein A chromatography. The liquid sample comprising the VH3-containing protein and at least one other substance may comprise host proteins (HCP), for example CHO cell, E. coli or yeast proteins. The content of CHO cell and E. coli proteins may be conveniently determined by immunoassays for those proteins, for example CHO HCP or E. coli HCP ELISA kits from Cygnus Technologies. The host cell proteins or CHO cell / E. coli proteins may be released during step b). Elution may be performed using any suitable solution used for elution from the Protein A medium. It may be, for example, a solution or buffer with a pH of 5 or less, such as a pH of 2.5-5 or 3-5. In some cases, it may also be a solution or buffer with a pH of 11 or more, such as a pH of 11-14 or 11-13. In some embodiments, the elution buffer or elution buffer gradient comprises at least one mono-, di- or trifunctional carboxylic acid or a salt of such a carboxylic acid. In certain embodiments, the elution buffer or elution buffer gradient comprises at least one anionic species selected from the group consisting of acetate, citrate, glycine, succinate, phosphate, and formate.

[0142] In some embodiments, the washing solution is alkaline, for example having a pH of 13 to 14. Such a solution provides efficient washing of the matrix, especially at the upper end of the interval.

[0143] In a particular embodiment, the washing solution comprises 0.1-2.0 M NaOH or KOH, such as 0.5-2.0 or 0.5-1.0 M NaOH or KOH. They are efficient washing solutions, especially when the concentration of NaOH or KOH is above 0.1 M or at least 0.5 M. The high stability of the polypeptides of the invention allows the use of such strongly alkaline solutions.

[0144] The method may also include a step of sanitizing the matrix with a sanitization liquid, which may include, for example, a peroxide, such as hydrogen peroxide, and / or a peracid, such as peracetic acid or performic acid.

[0145] In some embodiments, steps a)-d) are repeated at least 10 times, for example at least 50 times, 50-200 times, 50-300 times or 50-500 times. This is important for process economy, since the matrix can be reused many times. Steps a)-c) can also be repeated at least 10 times, for example at least 50 times, 50-200 times, 50-300 times or 50-500 times, and step d) is performed such that after multiple times of step c), step d) is performed at least 10 times, for example at least 50 times. Step d) can be performed, for example, every 2 to 20 times of step c).

[0146] The method of isolating immunoglobulins or fragments disclosed herein can be used for the isolation of fragments comprising at least one VH3 domain. Non-limiting examples of such fragments include Fab fragments, Fab' fragments, F(ab')2, scFab fragments, Fv fragments, scFv fragments, diabodies, triabodies and minibodies. The skilled artisan will be familiar with other fragments that may be isolated by the method. Thus, in one embodiment of the method, the fragment is selected from Fab fragments, Fab' fragments, F(ab')2, scFab fragments, Fv fragments, scFv fragments, diabodies, triabodies and minibodies.

[0147] In particular, the method may be suitable for the isolation of immunoglobulins or fragments thereof, where the immunoglobulins or fragments thereof are asymmetric immunoglobulins or fragments thereof. As used herein, the term "asymmetric immunoglobulins or fragments thereof" refers to immunoglobulins or fragments thereof in which two different heavy chains or fragments of heavy chains are paired. Such immunoglobulins or fragments may be bispecific. An illustrative example of an asymmetric immunoglobulin is the asymmetric bispecific antibody emicizumab (Chugai Pharmaceutical Co., Ltd.), which is used for the treatment of hemophilia A. Emicizumab has two heavy chains, one of VH3 class and one of VH1 class. The method allows for the efficient isolation of correctly paired variants (VH3-VH1) by using the VH3 interaction of one heavy chain. In this way, it is possible to separate homodimer (undesired VH3-VH3 and VH1-VH1) variants already in the capture step from heterodimer (desired VH3-VH1) variants, thus simplifying downstream processes. Without being bound by theory, it is assumed that the separation works on the following principle: homodimers (VH1-VH1) do not bind, so they enter the flow-through (FT); homodimers (VH3-VH3) bind more tightly compared to the correctly paired heterodimer VH1-VH3, since they bind through two interactions, thus creating an avidity effect. Therefore, correctly paired heterodimers are expected to elute under milder elution conditions, e.g. milder pH, than VH3-VH3 dimers. Thus, the resolution of VH1-VH3 and VH3-VH3 is achieved by one gradient elution or by two gradient elutions using step elutions with different pH. This is illustrated in the attached Example 6. In particular, the low or absent ability of the VH3 binding polypeptides and multimers thereof to bind to the Fc region of trastuzumab disclosed herein advantageously improves the purity of the isolated fractions, since any confounding Fc interactions are absent or very low.

[0148] Thus, in one embodiment of the method, the immunoglobulin or fragment thereof is an asymmetric immunoglobulin or fragment thereof. In particular, said asymmetric immunoglobulin or fragment thereof comprises a VH3 region. In one embodiment, said asymmetric immunoglobulin or fragment thereof is a multispecific immunoglobulin or fragment thereof, such as at least a bispecific or trispecific immunoglobulin or fragment thereof.

[0149] As the skilled person will appreciate, the conditions to which immunoglobulins and / or fragments thereof are subjected during isolation may affect their target binding capacity. It may therefore be advantageous for the elution step c) to be carried out under mild conditions, for example under mild pH conditions as exemplified in the enclosed Example 4. Thus, in one embodiment of said method, the separation matrix comprises a VH3 binding polypeptide according to SEQ ID NO: 14 or a multimer comprising SEQ ID NO: 14, and the pH level of elution is above 4, for example in the range of approximately 4.10 to 4.20, for example approximately 4.15. In another embodiment, said separation matrix comprises a VH3 binding polypeptide according to SEQ ID NO: 15 or a multimer comprising SEQ ID NO: 15, and the pH level of elution is below 4, for example in the range of approximately 3.7 to 3.80, for example approximately 3.75.

[0150] In one embodiment of the method described herein, when the method concerns the isolation of asymmetric immunoglobulins or fragments thereof, it is envisaged that the elution of the immunoglobulins or fragments thereof from the separation matrix is ​​by pH gradient or by stepwise elution at at least two different pH levels. It is understood that the method may involve the elution of both symmetric and asymmetric immunoglobulins or fragments thereof, or of either one or the other. Thus, in one embodiment: a) providing a liquid sample comprising a mixture of at least one symmetric immunoglobulin or fragment thereof and at least one asymmetric immunoglobulin or fragment thereof; b) contacting the liquid sample with a separation matrix, wherein the symmetric immunoglobulin or fragment thereof and the asymmetric immunoglobulin or fragment thereof bind to the separation matrix with different binding strengths, the separation matrix being defined as in claim 14; c) eluting said immunoglobulins or fragments thereof from the separation matrix, wherein said symmetric immunoglobulins or fragments thereof and said asymmetric immunoglobulins or fragments thereof are at least partially eluted separately from each other, and optionally d) washing the separation matrix with a washing solution The present invention provides a method for isolating an asymmetric immunoglobulin or a fragment thereof, comprising:

[0151] It is envisaged that the elution of the immunoglobulins or fragments thereof from the separation matrix, in which the symmetric immunoglobulins or fragments thereof and the asymmetric immunoglobulins or fragments thereof are at least partially eluted separately from each other, may be based on pH, ionic strength or a combination of both properties. The skilled person is familiar with different elution techniques. For example, the elution may be performed using a gradient or by stepwise change of the eluent.

[0152] In one embodiment, step c) comprises: c) eluting said immunoglobulins or fragments thereof from the separation matrix by a pH gradient or by stepwise elution at least two different pH levels, whereby said symmetric immunoglobulins or fragments thereof and said asymmetric immunoglobulins or fragments thereof are at least partially eluted at different pH levels. A method for engaging in the above is provided.

[0153] In one embodiment, the asymmetric immunoglobulin or fragment thereof comprises one VH3 domain. In one embodiment, the symmetric immunoglobulin or fragment thereof comprises at least two VH3 domains. In one embodiment, the symmetric immunoglobulin or fragment thereof comprises at least two VH3 domains or no VH3 domain. It should be understood that the VH3 domain is the VH3 domain of trastuzumab.

[0154] As explained above, the washing solution may be NaOH or KOH, e.g. 0.5 NaOH, or other suitable washing solutions for washing the separation matrix known in the art and discussed above. It is understood that the embodiments discussed above relating to, for example, elution buffers or gradients, washing solutions, optional matrix sanitizing steps, and the number of times steps a)-c) or a)-d) are repeated, are equally relevant to the method for isolation of asymmetric immunoglobulins or fragments thereof as to the method for isolation of immunoglobulins or fragments thereof, and will not be repeated here merely for the sake of brevity.

[0155] It should be understood that step c) may comprise the elution of one of the symmetric immunoglobulins or fragments thereof and the asymmetric immunoglobulins or fragments thereof, but not the other. Thus, in one embodiment of the method described herein, there is provided a method comprising a step c) comprising eluting at least one of the symmetric immunoglobulins or fragments thereof and the asymmetric immunoglobulins or fragments thereof at a pH level suitable for the elution of at least one of the symmetric immunoglobulins or fragments thereof and the asymmetric immunoglobulins or fragments thereof, but not the other, whereby the symmetric immunoglobulins or fragments thereof and the asymmetric immunoglobulins or fragments thereof are at least partially eluted at different pH levels.

[0156] In one embodiment of the method for isolating asymmetric immunoglobulins or fragments thereof, said separation matrix is ​​as defined herein. As explained above, the method allows for the efficient separation of immunoglobulins or fragments thereof that are paired in an undesired manner (e.g. homomeric, also called symmetric) from immunoglobulins or fragments thereof that are paired in a desired manner (e.g. heteromeric, also called asymmetric).

[0157] It should be understood that the properties of the resin used may affect the efficiency of separation of said immunoglobulins or fragments thereof. For example, resolution may be improved using solid supports characterized by small particles or beads, such as agarose beads. Thus, in one embodiment, the separation matrix comprises a solid support with a particle or bead size of at most 75 μm, such as at most 60 μm, such as at most 50 μm or less. In one embodiment, said particle or bead size is the diameter of the particle or bead. In one embodiment, said beads are agarose beads.

[0158] In a particular embodiment, when a method for isolating asymmetric immunoglobulins or fragments thereof as disclosed herein is provided, 25% or less, such as 20% or less, such as 15% or less, such as 10% or less of asymmetric immunoglobulins or fragments thereof are present in the same eluate as symmetric immunoglobulins or fragments thereof. In a particular embodiment, when a method for isolating asymmetric immunoglobulins or fragments thereof as disclosed herein is provided, 25% or less, such as 20% or less, such as 15% or less, such as 10% or less, such as 5% or less, such as 3% or less of the eluate contains both symmetric and asymmetric immunoglobulins or fragments thereof. For clarity, in this context, the eluate should be interpreted as the fraction containing a significant amount of protein, which protein bound to the separation matrix after contacting the protein sample with the separation matrix as disclosed herein. The other fraction is considered as the flow-through.

[0159] In one embodiment, a method is provided for isolating an asymmetric immunoglobulin or a fragment thereof, wherein the asymmetric immunoglobulin or a fragment thereof is a multispecific immunoglobulin or a fragment thereof, such as at least a bispecific or trispecific immunoglobulin or a fragment thereof.

[0160] The terms "VH3 binding polypeptide" and "VH3 binding protein" mean a polypeptide or protein, respectively, capable of binding to VH3 of an antibody and include, for example, Protein A, or any fragment or fusion protein thereof which maintains said binding properties.

[0161] The term "linker" as used herein refers to an element that connects two polypeptide units, monomers or domains together in a multimer.

[0162] The term "spacer" as used herein refers to an element that connects a polypeptide or polypeptide multimer to a support.

[0163] The term "dynamic binding capacity (DBC)" of a chromatography column in the context of protein purification describes the maximum amount of target protein that can be loaded onto the column without unnecessary losses, measured under realistic experimental conditions (defined flow rate, real protein sample).

[0164] Although the present invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will recognize that various changes can be made and equivalents can be substituted for the elements thereof without departing from the true scope of the invention. In addition, many modifications can be made to adapt a particular situation or molecule to the teachings of the invention without departing from its essential scope. Therefore, it is not intended that the invention be limited to any particular embodiment contemplated, but rather that the invention includes all embodiments falling within the scope of the appended claims. [Brief description of the drawings]

[0165] [Figure 1A]Figure 1 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NOs: 1-4) using 5000 nM Trastuzumab Fc fragment as analyte, compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of Trastuzumab, and a non-Fc binding control (SEQ ID NO: 180). For clarity of presentation, the results of the Biacore analysis are shown in Figure 1A in two graphs, one including and one not including the data corresponding to SEQ ID NO: 88. [Figure 1B] Figure 1 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NOs: 1-4) compared to a non-Fc binding control (SEQ ID NO: 180) using 5000 nM trastuzumab Fab fragment as analyte. [Figure 1C] FIG. 1 is a plot of an example of alkaline wash stability assessed using Biacore™ of tested VH3 binding polypeptides of the invention (SEQ ID NOs: 1-4) compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, and a non-Fc binding control (negative CTRL) with no alkaline wash stability. [Figure 2A] Figure 2 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NOs: 5-12) using 5000 nM Trastuzumab Fc fragment as analyte, compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of Trastuzumab. For clarity of presentation, the results of the Biacore analysis are shown in Figure 2A in two graphs, one including and one not including the data corresponding to SEQ ID NO: 88. [Figure 2B] Figure 2 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NOs: 5-12) using 625 nM trastuzumab as analyte, compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab. For clarity of presentation, the results of the Biacore analysis are shown in Figure 2B in two graphs, one including and one not including the data corresponding to SEQ ID NO: 88. [Figure 2C]FIG. 1 is an example plot of the alkaline wash stability of tested VH3 binding polypeptides of the invention (SEQ ID NOs: 5-12) compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, and a non-Fc binding control (negative CTRL) that has no alkaline wash stability, assessed using Biacore™. [Figure 3A] Figure 3 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NOs: 13-15) using 5000 nM Trastuzumab Fc fragment as analyte, compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of Trastuzumab. For clear presentation, the results of the Biacore analysis are shown in Figure 3A in two graphs, one including and one not including the data corresponding to SEQ ID NO: 88. [Figure 3B] 1 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NO: 10 and SEQ ID NOs: 13-15) using 2500 nM trastuzumab Fab fragment as analyte, compared to an alkaline wash stable positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, and a non-Fc binding control (negative CTR) that is not alkaline wash stable. [Figure 3C] 1 shows a Biacore analysis of VH3 binding polypeptides of the invention (SEQ ID NO: 10 and SEQ ID NOs: 13-15) using 625 nM trastuzumab as analyte, compared to an alkaline wash stable positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, and a non-Fc binding control with no alkaline wash stability (negative CTR). [Figure 3D] FIG. 1 is a plot of an example of alkaline wash stability assessed using Biacore™ of tested VH3 binding polypeptides of the invention (SEQ ID NO: 10 and SEQ ID NOs: 13-15) compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, and a non-Fc binding control with no alkaline wash stability (negative CTRL). [Figure 4A]FIG. 1 shows the elution profile of emicizumab feed that also contains VH1 / VH3 bispecific antibody and VH3 / VH3 mismatched homodimer using a pH gradient and a control resin (SEQ ID NO: 181). [Figure 4B] FIG. 1 shows the elution profile of emicizumab feed, also including VH1 / VH3 bispecific antibody and VH3 / VH3 mismatched homodimer, using a pH gradient and a resin according to the invention in which SEQ ID NO: 177 is coupled to a 60 μm agarose-based matrix. [Figure 4C] FIG. 1 shows the elution profile of emicizumab feed, also including VH1 / VH3 bispecific antibody and VH3 / VH3 mismatched homodimer, using a pH gradient and a resin according to the invention in which SEQ ID NO: 177 is coupled to a 50 μm agarose-based matrix. [Diagram 5] 1 is a table of amino acid sequences of examples of VH3 binding polypeptides of the disclosure (SEQ ID NOs: 1-82) and derivatives of domains A, B, C, D, and E of Staphylococcal protein A (SEQ ID NOs: 83-87). In the VH3 binding polypeptides of the disclosure, predicted sequence A spans from residue 8 to residue 36 in each of SEQ ID NOs: 1-82. Amino acid sequence B spans from residues 37-54, and binding module sequence C, which is predicted to constitute a complete three-helix bundle within each of these VH3 binding polypeptides, spans from residue 8 to residue 54 of the 58-mer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0166] Overview of Examples 1 to 10 The following examples disclose the surprising identification of novel VH3-binding SpA-derived polypeptides that exhibit no or significantly reduced affinity for the Fc region of immunoglobulins and are alkaline wash stable.

[0167] A class of alkaline wash stable VH3 binding polypeptides that exhibit loss of trastuzumab Fc binding was identified (SEQ ID NOs: 1-4). The VH3 binding polypeptides (Fr1-4; SEQ ID NOs: 1-4) were evaluated in the following aspects: trastuzumab Fc binding, trastuzumab Fab binding and alkaline wash stability (Example 1).

[0168] Further alkaline wash stable VH3 binding polypeptides were identified (Fr5-12; SEQ ID NOs: 5-12) that do not show binding to the Fc fragment of trastuzumab. The VH3 binding polypeptides (Fr5-12; SEQ ID NOs: 5-12) were evaluated in the following aspects: trastuzumab Fc binding, trastuzumab binding and alkaline wash stability (Example 2).

[0169] Based on the VH3 binding polypeptide with improved alkaline wash stability (SEQ ID NO: 10), a further set of alkaline wash stable VH3 binding polypeptides was identified (Fr31-33; SEQ ID NOs: 13-15) that did not exhibit trastuzumab Fc binding and exhibited improved binding affinity to trastuzumab and Fab fragments of trastuzumab. The VH3 binding polypeptides (SEQ ID NOs: 13-15) were evaluated in the following aspects: trastuzumab Fc binding, trastuzumab Fab binding, trastuzumab binding and alkaline wash stability (Example 3).

[0170] A subset of alkaline wash stable VH3 binding polypeptides in which Fc binding was lost (Fr32 and Fr33; SEQ ID NOs: 14 and 15) was selected for further testing on the column. Further testing included multimerization of the polypeptides into tetramers and hexamers and expression of the multimers in E. coli. Primary characterization was performed on these multimeric VH3 binding polypeptides (Fr32-4, Fr33-4 and Fr33-6; SEQ ID NOs: 175-177) to evaluate the alkaline wash stable VH3 binding polypeptide multimers in terms of dynamic binding capacity (DBC), pH elution and alkaline stability (Example 4).

[0171] The VH3 binding polypeptide multimeric variant (Fr33-6; SEQ ID NO: 177) was selected for further testing to evaluate its binding specificity to different VH1 and VH3 class target molecules (Examples 5 and 6) and to evaluate its binding specificity to a VH1 / VH3 asymmetric bispecific antibody (Examples 6 and 7).

[0172] The effects of mutations at positions 9, 10, 11, 13, 14, 17, 18, 28 and 29 were evaluated with different scaffolds (SEQ ID NOs: 89-94) in the following aspects: trastuzumab Fc binding, trastuzumab Fab binding, trastuzumab binding and alkaline washing stability (Examples 8 and 9).

[0173] Based on the sequence of Fr33 (SEQ ID NOs: 15 and 56), further novel classes of VH3 binding polypeptides were created (SEQ ID NOs: 16-41 and SEQ ID NOs: 57-82) with amino acid variations at positions 13, 14 and 26. The VH3 binding polypeptides (SEQ ID NOs: 16-41 and SEQ ID NOs: 57-82) were evaluated in the following aspects: trastuzumab Fc binding, trastuzumab Fab binding, trastuzumab binding and alkaline wash stability (Example 10).

[0174] Example 1 Biacore evaluation of VH3 binding polypeptides overview The effect of mutations was investigated to identify amino acid positions that allow reduced binding to the Fc region while retaining alkaline wash stability. A class of VH3 binding polypeptides derived from SEQ ID NO:88 (Fr1-4, SEQ ID NO:1-4) were identified as shown in Table 1. The polypeptides were evaluated in the following aspects: Trastuzumab Fc binding, Trastuzumab Fab binding and alkaline wash stability.

[0175] [Table 1]

[0176] Polypeptide variants (SEQ ID NOs: 1-4) were evaluated along with an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, a non-Fc binding control (SEQ ID NO: 180), and a non-Fc binding control with no alkaline wash stability (negative CTRL).

[0177] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of Fab interactions (tested by injecting high concentrations of Fab fragments generated from trastuzumab) (3) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) was evaluated.

[0178] The present inventors have surprisingly discovered a class of novel VH3-binding SpA-derived polypeptides (SEQ ID NOs: 1 to 4) that have amino acid diversity at positions 9, 10, 11, 13, 14, and optionally 17 and 18, which exhibit no affinity or significantly reduced affinity for the Fc region of trastuzumab without losing alkaline washing stability.

[0179] Materials and Methods Generation of His-purified candidate polypeptides Materials and equipment used were as follows: glycerol stocks of SEQ ID NOs: 1-4; LB culture medium, 2YT culture medium, carbenicillin, IPTG 1M, 125 ml and 250 ml baffled glass shake flasks, Infors HT shaker incubator; His-GraviTrap™ kit (Cytiva™), PD10 GraviTraps (Cytiva™).

[0180] 5 μl of glycerol stock of each variant was inoculated into 4 ml of LB supplemented with 200 μg / ml carbenicillin in a 125 ml flask and incubated overnight at 37° C. at 200 rpm.

[0181] Protein expression culture medium (2YT medium supplemented with 100 μg / ml carbenicillin) was prepared and added to fill baffled shake flasks (50 ml per flask, 20 flasks in total). Each flask was inoculated with approximately 500 μl from the overnight culture to obtain a starting OD600 of 0.05. The flasks were incubated in an Infors HT shaker incubator at 37° C. and 130 rpm shaking for approximately 2.5 hours until the OD600 reached 0.7. Then 25 μl of IPTG (1M) was added and the flasks were incubated overnight in an Infors HT shaker incubator at 27° C. and 130 rpm shaking before pelleting (8000 g, 5 min) and resuspending the culture in 10 ml of PBS + 20 mM imidazole in a Falcon tube. To generate crude Varian lysate, the Falcon tube was incubated in a water bath at 80-85°C for 10 min, followed by pelleting of cell debris by centrifugation at 10000g for 10 min and filtering the supernatant through a 0.2 μm filter.

[0182] Samples were purified using the His GraviTraps kit according to the kit instructions (PBS+20mM imidazole was used as binding / wash buffer). Buffer exchange into PBS was performed using PD10 Gravitraps and kit instructions.

[0183] The concentration of the buffer exchanged samples was measured using a NanoDrop (Thermo Scientific) according to the manufacturer's instructions. Following purification, samples were kept in the refrigerator until all measurements were performed, then transferred to Eppendorf tubes and stored in the refrigerator.

[0184] Biacore analysis of binding affinity of trastuzumab to Fc and Fab fragments To assess binding to Fc (SEQ ID NO: 184) and Fab (SEQ ID NOs: 182 and 183), VH3 binding polypeptides (SEQ ID NOs: 1-4) were immobilized on a Biacore CM5 chip. The Fc fragment of trastuzumab (5000 nM) and the Fab fragment of trastuzumab (5000 nM) were used as analytes.

[0185] pH study on immobilization conditions: Materials and instruments used were as follows: VH3 binding polypeptide variants (SEQ ID NOs: 1-4); Biacore™ CM5 chips (Cytiva™); Biacore™ acetate buffers with different pH (Cytiva™); Biacore™ 8K+ instrument (Cytiva™).

[0186] The pH study was carried out on a Biacore™ 8K+ using standard methods defined in the software at different pH values ​​(pH 4.5, 5, 5.5). The pH value corresponding to one unit below the pI of the target ligand was found to result in maximum surface attraction.

[0187] Biacore binding analysis: Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), VH3 binding polypeptide (SEQ ID NOs: 1-4); Biacore™ 8K+ (Cytiva™); Fab (prepared from in-house trastuzumab), Fc from trastuzumab.

[0188] Immobilization was performed by coupling of VH3 binding polypeptide variants in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1) using standard methods in the Biacore software. VH3 binding polypeptide variants were diluted to the appropriate pH (based on pH studies) in acetate buffer in the concentration range of 10-30 μg / ml. Immobilization levels were somewhat different between the different polypeptide variants (approximately 600-1300 Ru).

[0189] In each run, the polypeptide was immobilized in FC2. Multiple chips were used until all candidates were tested. Biacore™ methods for binding analysis: Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection: 600s / 10min on both flow cells (FC1 and FC2) Dissociation time: 600s / 10min Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0190] The injections (cycles) of analyte (Fab or Fc fragment of trastuzumab) in each channel were as follows: buffer, 156 nM, 313 nM, 625 nM, 1250 nM, 2500 nM, 5000 nM.

[0191] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0192] Biacore analysis of alkaline washing stability Alkaline wash stability was assessed by immobilizing VH3 binding polypeptides (SEQ ID NOs: 1-4) on a CM5 Biacore chip followed by 100 repeated cycles of binding to trastuzumab (1 μM) followed by injection of NaOH (0.5 M).

[0193] Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), VH3 binding polypeptide (SEQ ID NOs: 1-4); Biacore™ 8K+ (Cytiva™); trastuzumab; 0.5 M NaOH.

[0194] Immobilization was performed by coupling of VH3 binding polypeptide variants in FC2 and activation / inactivation in FC1 using standard methods in the Biacore software. VH3 binding polypeptide variants were diluted to the appropriate pH (based on pH studies) in acetate buffer in the concentration range of 10-30 μg / ml. The immobilization levels were somewhat different between the different polypeptide variants (approximately 600-1300 Ru). Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection 1 (Trastuzumab 1 μM): 300 s on both flow cells Dissociation time 1:60s Sample injection 2 (0.5 M NaOH): 600 s on both flow cells Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0195] This cycle was repeated over 100 times to follow the stability of the trastuzumab response values.

[0196] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0197] result Biacore analysis of binding affinity of trastuzumab to Fc and Fab fragments The response levels of the VH3 binding polypeptide variants' interactions with the Fc and Fab of trastuzumab are depicted in Figures 1A and 1B. The data show that the VH3 binding polypeptide variants (SEQ ID NOs: 1-4) exhibit significantly reduced or abolished binding affinity to the Fc fragment of trastuzumab while retaining binding to the Fab fragment of trastuzumab. The positive control (SEQ ID NO: 88) exhibits binding to the Fc fragment of trastuzumab of 2000 RU upon injection. The non-Fc binding control (SEQ ID NO: 180) shows no interaction with Fc (0 RU upon injection) and weak binding to Fab of approximately 100 RU upon injection.

[0198] Biacore analysis of alkaline washing stability Alkaline wash stability was visualized by plotting normalized trastuzumab response values ​​(% of response at cycle 2) from the second cycle to the final cycle run described above. An alkaline wash stability positive control (SEQ ID NO: 88) that binds both Fc and Fab fragments, and a non-Fc binding control with no alkaline wash stability (negative CTRL) were presented as controls in each of the separate stability runs. Trastuzumab response values ​​after 100 wash cycles are summarized in Table 2. Figure 1C shows a plot of alkaline wash stability of the tested VH3 binding polypeptide variants (SEQ ID NOs: 1-4) compared to SEQ ID NO: 88 and a non-alkaline wash stable non-Fc binding control (negative CTRL). In each cycle, 0.5 M NaOH was injected at 10 μl / min with a contact time of 10 min.

[0199] [Table 2]

[0200] conclusion Novel VH3-binding polypeptides (SEQ ID NOs: 1 to 4) were identified that did not exhibit binding to the Fc region of trastuzumab but exhibited binding to the Fab fragment of trastuzumab.

[0201] SEQ ID NOs: 1-4 are alkaline wash stable and retain at least 21.5% of their trastuzumab response (% bound) in cycle 2 after 100 wash cycles with 10 μl / min 0.5 M NaOH with a 10 minute contact time.

[0202] It is concluded that said combination of at least amino acids at positions 9, 10, 11, 13, 14 and optionally 17 and 18 as shown in Table 1 provides advantageous properties for a VH3 binding polypeptide according to the invention, such as alkaline wash stability, at least retaining binding to the Fab fragment of trastuzumab and exhibiting reduced or abolished binding to the Fc fragment of trastuzumab.

[0203] Example 2 Biacore evaluation of VH3 binding polypeptides with improved alkaline wash stability overview Based on the results of the Biacore evaluation of the VH3 binding polypeptides (SEQ ID NOs: 1-4) shown in Example 1, eight further VH3 binding polypeptides (Fr7-14; SEQ ID NOs: 5-12) were identified with amino acid diversity at seven positions (9, 10, 11, 13, 14, 17 and 18) as shown in Table 3. The polypeptides were evaluated in the following aspects: trastuzumab Fc binding, trastuzumab binding and alkaline wash stability.

[0204] [Table 3]

[0205] Polypeptide variants (SEQ ID NOs: 5-12) were evaluated along with an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab, and a non-Fc binding control (negative CTRL) that has no alkaline wash stability.

[0206] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of trastuzumab interaction (tested by injecting high concentrations of trastuzumab). (3) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) was evaluated.

[0207] The present inventors have surprisingly found further novel VH3-binding polypeptides (SEQ ID NOs: 5 to 12) having amino acid variability at positions 9, 10, 11, 13, 14, and optionally 17 and 18, which exhibit binding to trastuzumab, significantly reduced or absent affinity for the Fc region of trastuzumab, and are alkaline wash stable.

[0208] Materials and Methods Generation of His-purified candidate polypeptides Materials and equipment used were as follows: glycerol stocks of SEQ ID NOs: 5-12; LB culture medium, 2YT culture medium, carbenicillin, IPTG 1M, 125 ml and 250 ml baffled glass shake flasks, Infors HT shaker incubator; His-GraviTrap™ Kit (Cytiva™), PD10 GraviTraps (Cytiva™).

[0209] 5 μl of glycerol stock of each variant was inoculated into 4 ml of LB supplemented with 200 μg / ml carbenicillin in a 125 ml flask and incubated overnight at 37° C. at 200 rpm.

[0210] Protein expression culture medium (2YT medium supplemented with 100 μg / ml carbenicillin) was prepared and added to fill baffled shake flasks (50 ml per flask, 20 flasks in total). Each flask was inoculated with approximately 500 μl from the overnight culture to obtain a starting OD600 of 0.05. The flasks were incubated in an Infors HT shaker incubator at 37° C. and 130 rpm shaking for approximately 2.5 hours until the OD600 reached 0.7. Then 25 μl of IPTG (1M) was added and the flasks were incubated overnight in an Infors HT shaker incubator at 27° C. and 130 rpm shaking before pelleting (8000 g, 5 min) and resuspending the culture in 10 ml of PBS + 20 mM imidazole in a Falcon tube. To generate crude Varian lysate, the Falcon tube was incubated in a water bath at 80-85°C for 10 min, followed by pelleting of cell debris by centrifugation at 10000g for 10 min and filtering the supernatant through a 0.2 μm filter.

[0211] Samples were purified using the His GraviTraps kit according to the kit instructions (PBS+20mM imidazole was used as binding / wash buffer). Buffer exchange into PBS was performed using PD10 Gravitraps and kit instructions.

[0212] The concentration of the buffer exchanged samples was measured using a NanoDrop (Thermo Scientific) according to the manufacturer's instructions. Following purification, samples were kept in the refrigerator until all measurements were performed, then transferred to Eppendorf tubes and stored in the refrigerator.

[0213] Biacore analysis of binding affinity to Fc fragment of trastuzumab and trastuzumab To assess binding to the Fc fragment of trastuzumab and trastuzumab, VH3 binding polypeptides were immobilized on a Biacore CM5 chip. The Fc fragment of trastuzumab (5000 nM) and trastuzumab (625 nM) were used as analytes.

[0214] pH study on immobilization conditions: Materials and instruments used were as follows: VH3 binding polypeptide variants (SEQ ID NOs: 5-12); Biacore CM5 chips (Cytiva™); Biacore acetate buffers with different pH (Cytiva™); Biacore™ 8K+ instrument (Cytiva™).

[0215] The pH study was carried out on a Biacore™ 8K+ using standard methods defined in the software at different pH values ​​(pH 4.5, 5, 5.5). The pH value corresponding to one unit below the pI of the target ligand was found to result in maximum surface attraction.

[0216] Biacore binding analysis: Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), VH3 binding polypeptide (SEQ ID NOs: 5-12); Biacore™ 8K+ (Cytiva™); Fc from trastuzumab (SEQ ID NO: 184); trastuzumab (SEQ ID NOs: 178 and 179).

[0217] Immobilization was performed by coupling of VH3 binding polypeptide variants in FC2 and activation / inactivation in FC1 using standard methods in the Biacore software. VH3 binding polypeptide variants were diluted to the appropriate pH (based on pH studies) in acetate buffer in the concentration range of 10-30 μg / ml. The immobilization levels were somewhat different between the different polypeptide variants (approximately 600-1300 Ru).

[0218] In each run, the polypeptide was immobilized in FC2. Multiple chips were used until all candidates were tested. Biacore methods for binding analysis: Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection: 600s / 10min on both flow cells (FC1 and FC2) Dissociation time: 600s / 10min Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0219] The injections (cycles) of analyte (trastuzumab or Fc fragment of trastuzumab) in each channel were as follows: buffer, 156 nM, 313 nM, 625 nM, 1250 nM, 2500 nM, 5000 nM.

[0220] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0221] Biacore analysis of alkaline washing stability Alkaline wash stability was assessed by immobilizing VH3 binding polypeptides (SEQ ID NOs: 5-12) on a CM5 Biacore™ chip followed by 100 repeated cycles of binding to trastuzumab (1 μM) followed by injection of NaOH (0.5 M).

[0222] Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), VH3 binding polypeptide (SEQ ID NOs: 5-12); Biacore™ 8K+ (Cytiva™); trastuzumab; 0.5 M NaOH.

[0223] Immobilization was performed by coupling of VH3 binding polypeptide variants in FC2 and activation / inactivation in FC1 using standard methods in the Biacore software. VH3 binding polypeptide variants were diluted to the appropriate pH (based on pH studies) in acetate buffer in the concentration range of 10-30 μg / ml. The immobilization levels were somewhat different between the different polypeptide variants (approximately 600-1300 Ru). Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection 1 (Trastuzumab 1 μM): 300 s on both flow cells Dissociation time 1:60s Sample injection 2 (0.5 M NaOH): 600 s on both flow cells Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0224] This cycle was repeated over 100 times to track the stability of the trastuzumab response values.

[0225] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0226] result Biacore analysis of binding affinity to Fc fragment of trastuzumab and trastuzumab The response levels of the interaction of the VH3 binding polypeptide variants (SEQ ID NOs: 5-12) with the Fc fragment of trastuzumab and trastuzumab are depicted in Figures 2A and 2B. The data show that the novel VH3 binding polypeptide variants (SEQ ID NOs: 5-12) exhibit significantly reduced or abolished binding affinity to the Fc fragment of trastuzumab while retaining binding to trastuzumab. The positive control (SEQ ID NO: 88) shows binding of >2000 RU and >6000 RU for both Fc and trastuzumab with each injection.

[0227] Biacore analysis of alkaline washing stability Alkaline wash stability was visualized by plotting normalized trastuzumab response values ​​(% of response at cycle 2) from the second cycle to the final cycle run described above. An alkaline wash stability positive control (SEQ ID NO: 88) that binds both Fc and Fab fragments, and a non-Fc binding control without alkaline wash stability (negative CTRL) were presented as controls in each of the separate stability runs. Trastuzumab response values ​​after 100 wash cycles are summarized in Table 4. Figure 2C shows a plot of alkaline wash stability of the tested VH3 binding polypeptide variants (SEQ ID NOs: 5-12) compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both Fc and Fab fragments, and a non-Fc binding control without alkaline wash stability (negative CTRL). In each cycle, 0.5 M NaOH was injected at 10 μl / min with a contact time of 10 min.

[0228] [Table 4]

[0229] conclusion Further novel VH3 binding polypeptides (SEQ ID NOs: 5-12) have been identified that exhibit binding to trastuzumab but not to the Fc fragment of trastuzumab.

[0230] SEQ ID NOs:5-12 are alkaline wash stable and retain at least 26% of their trastuzumab response (% bound) in cycle 2 after 100 wash cycles with 10 μl / min 0.5 M NaOH with a 10 minute contact time.

[0231] It is concluded that said combinations of amino acids at at least positions 9, 10, 11, 13, 14 and optionally positions 17 and 18, as shown in Table 3, result in the advantageous properties of the VH3 binding polypeptides according to the invention herein, SEQ ID NOs: 5-12 being alkaline wash stable, retaining binding to trastuzumab and showing no binding to the Fc fragment of trastuzumab.

[0232] By using information from alkaline wash stability assays together with amino acid sequence information, X9X 10 X 11 -X 13 X 14 Scaffolds with motifs AQR-LA and X9X 10 X 11 -X 13 X 14 -X 17 X 18 It is concluded that scaffolds with the motif AYR-LA-AR at position X9X show particularly desirable alkaline washing stability. 10 X 11 -X 13 X 14 -X 17 X 18 VH3 binding polypeptides comprising a scaffold with the motif AYR-LA--AR at position 1 have the further advantageous property that the presence of a tyrosine (Y at position 10) in the sequence allows the measurement of the concentration of said VH3 binding polypeptide by spectrophotometry.

[0233] Example 3 Biacore evaluation of VH3 binding polypeptides with improved binding affinity to the Fab of trastuzumab overview Based on the results of the Biacore evaluation of the further VH3 binding polypeptides (SEQ ID NOs: 5-12) shown in Example 2, three further VH3 binding polypeptide variants (Fr31-33; SEQ ID NOs: 13-15) were identified with amino acid diversity at nine positions (9, 10, 11, 13, 14, 17, 18, 28 and 29) as shown in Table 5. The polypeptides were evaluated in the following aspects: Trastuzumab Fc binding, Trastuzumab Fab binding, Trastuzumab binding and alkaline wash stability.

[0234] [Table 5]

[0235] Polypeptide variants (SEQ ID NOs: 13-15) were evaluated along with an alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc and Fab fragments, and a non-Fc binding control (negative CTRL) that has no alkaline wash stability.

[0236] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of Fab interactions (tested by injecting high concentrations of Fab fragments generated from trastuzumab) (3) Affinity assessment of trastuzumab interaction (tested by injecting high concentrations of trastuzumab). (4) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) was evaluated.

[0237] The present inventors have surprisingly found further novel VH3-binding polypeptides (SEQ ID NOs: 13-15) having amino acid diversity at nine positions (9, 10, 11, 13, 14, 17, 18, 28 and 29) which exhibit the even more desirable properties of being alkaline washing stable and exhibiting binding to trastuzumab and the Fab fragment of trastuzumab, but no affinity for the Fc region of trastuzumab.

[0238] Materials and Methods Generation of His-purified candidate polypeptides Materials and equipment used were as follows: glycerol stocks of SEQ ID NOs: 13-15; LB culture medium, 2YT culture medium, carbenicillin, IPTG 1M, 125 ml and 250 ml baffled glass shake flasks, Infors HT shaker incubator; His-GraviTrap™ Kit (Cytiva™), PD10 GraviTraps (Cytiva™).

[0239] 5 μl of glycerol stock of each variant was inoculated into 4 ml of LB supplemented with 200 μg / ml carbenicillin in a 125 ml flask and incubated overnight at 37° C. at 200 rpm.

[0240] Protein expression culture medium (2YT medium supplemented with 100 μg / ml carbenicillin) was prepared and added to fill baffled shake flasks (50 ml per flask, 20 flasks in total). Each flask was inoculated with approximately 500 μl from the overnight culture to obtain a starting OD600 of 0.05. The flasks were incubated in an Infors HT shaker incubator at 37° C. and 130 rpm shaking for approximately 2.5 hours until the OD600 reached 0.7. Then 25 μl of IPTG (1M) was added and the flasks were incubated overnight in an Infors HT shaker incubator at 27° C. and 130 rpm shaking before pelleting (8000 g, 5 min) and resuspending the culture in 10 ml of PBS + 20 mM imidazole in a Falcon tube. To generate crude Varian lysate, the Falcon tube was incubated in a water bath at 80-85°C for 10 min, followed by pelleting of cell debris by centrifugation at 10000g for 10 min and filtering the supernatant through a 0.2 μm filter.

[0241] Samples were purified using the His GraviTraps kit according to the kit instructions (PBS+20mM imidazole was used as binding / wash buffer). Buffer exchange into PBS was performed using PD10 Gravitraps and kit instructions.

[0242] The concentration of the buffer exchanged samples was measured using a NanoDrop (Thermo Scientific) according to the manufacturer's instructions. Following purification, samples were kept in the refrigerator until all measurements were performed, then transferred to Eppendorf tubes and stored in the refrigerator.

[0243] Biacore analysis of the Fc and Fab fragments of trastuzumab and their binding affinity to trastuzumab To evaluate binding to trastuzumab, and the Fc and Fab fragments of trastuzumab, the VH3 binding polypeptides (SEQ ID NOs: 13-15) were immobilized on a Biacore™ CM5 chip. The Fc fragment of trastuzumab (5000 nM), the Fab fragment of trastuzumab (2500 nM), and trastuzumab (625 nM) were used as analytes.

[0244] pH study on immobilization conditions: Materials and instruments used were as follows: VH3 binding polypeptide variants (SEQ ID NOs: 13-15); Biacore™ CM5 chips (Cytiva™); Biacore acetate buffers with different pH (Cytiva™); Biacore™ 8K+ instrument (Cytiva™).

[0245] The pH study was carried out on a Biacore™ 8K+ using standard methods defined in the software at different pH values ​​(pH 4.5, 5, 5.5). The pH value corresponding to one unit below the pI of the target ligand was found to result in maximum surface attraction.

[0246] Biacore binding analysis: Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), VH3 binding polypeptide (SEQ ID NOs: 13-15); Biacore™ 8K+ (Cytiva™); Fab (prepared from in-house trastuzumab), Fc from trastuzumab, trastuzumab.

[0247] Immobilization was performed by coupling of VH3 binding polypeptide variants in FC2 and activation / inactivation in FC1 using standard methods in the Biacore software. VH3 binding polypeptide variants were diluted to the appropriate pH (based on pH studies) in acetate buffer in the concentration range of 10-30 μg / ml. The immobilization levels were somewhat different between the different polypeptide variants (approximately 600-1300 Ru).

[0248] In each run, the polypeptide was immobilized in FC2. Multiple chips were used until all candidates were tested. Biacore methods for binding analysis: Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection: 600s / 10min on both flow cells (FC1 and FC2) Dissociation time: 600s / 10min Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0249] The injections (cycles) of analyte (Fab or Fc fragment of trastuzumab, or trastuzumab) in each channel were as follows: buffer, 156 nM, 313 nM, 625 nM, 1250 nM, 2500 nM, 5000 nM.

[0250] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0251] Biacore analysis of alkaline washing stability Alkaline wash stability was assessed by immobilizing VH3 binding polypeptides (SEQ ID NOs: 13-15) on a CM5 Biacore chip followed by 100 repeated cycles of binding to trastuzumab (1 μM) followed by injection of NaOH (0.5 M).

[0252] Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), VH3 binding polypeptide (SEQ ID NOs: 13-15); Biacore™ 8K+ (Cytiva™); trastuzumab; 0.5 M NaOH.

[0253] Immobilization was performed by coupling of VH3 binding polypeptide variants in FC2 and activation / inactivation in FC1 using standard methods in the Biacore software. VH3 binding polypeptide variants were diluted to the appropriate pH (based on pH studies) in acetate buffer in the concentration range of 10-30 μg / ml. The immobilization levels were somewhat different between the different polypeptide variants (approximately 600-1300 Ru). Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection 1 (Trastuzumab 1 μM): 300 s on both flow cells Dissociation time 1:60s Sample injection 2 (0.5 M NaOH): 600 s on both flow cells Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0254] This cycle was repeated over 100 times to track the stability of the trastuzumab response values.

[0255] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0256] result Biacore analysis of the Fc and Fab fragments of trastuzumab and their binding affinity to trastuzumab The response levels of the interaction of Trastuzumab Fc, Trastuzumab Fab and Trastuzumab with the VH3 binding polypeptide variants (SEQ ID NOs: 13-15) are depicted in Figures 3A, 3B and 3C. The results show that the VH3 binding polypeptide variants (SEQ ID NOs: 13-15) exhibit significantly reduced or abolished binding affinity to the Trastuzumab Fc fragment while retaining binding to Trastuzumab and the Trastuzumab Fab fragment. The alkaline wash stability positive control (SEQ ID NO: 88), which binds both the Trastuzumab Fc fragment and the Trastuzumab Fab fragment, exhibits >2000 RU, >300 RU and >6000 RU of binding to Trastuzumab Fc, Trastuzumab Fab and Trastuzumab upon injection, respectively.

[0257] Three additional VH3 binding polypeptides (SEQ ID NO: 13-15) show improved binding to trastuzumab and trastuzumab Fab compared to the VH3 binding polypeptide tested in Example 2 (SEQ ID NO: 10). SEQ ID NO: 13 and SEQ ID NO: 15 show improved binding to both Fab and trastuzumab compared to a non-Fc binding control (negative CTRL) with no alkaline wash stability. SEQ ID NO: 13 and SEQ ID NO: 15 show improved binding to Fab compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both Fc and Fab fragments. SEQ ID NO: 13 and SEQ ID NO: 15 show trastuzumab response values ​​comparable to the positive control SEQ ID NO: 88.

[0258] Biacore analysis of alkaline washing stability Stability was visualized by plotting normalized trastuzumab response values ​​(% of response at cycle 2) from the second cycle to the final cycle run described above. An alkaline wash stability positive control (SEQ ID NO: 88) that binds both Fc and Fab fragments, and a non-Fc binding control without alkaline wash stability (negative CTRL) were presented as controls in each of the separate stability runs. Trastuzumab response values ​​after 100 wash cycles are summarized in Table 6. Figure 3D shows a plot of alkaline wash stability of the tested VH3 binding polypeptide variants (SEQ ID NOs: 13-15) compared to an alkaline wash stability positive control (SEQ ID NO: 88) that binds both Fc and Fab fragments of trastuzumab, and a non-Fc binding control without alkaline wash stability (negative CTRL). In each cycle, 0.5 M NaOH was injected at 10 μl / min with a contact time of 10 min.

[0259] SEQ ID NO:14 has alkaline wash stability comparable to the VH3 binding polypeptide (SEQ ID NO:10) tested in Example 2. SEQ ID NO:13 and SEQ ID NO:15 have improved alkaline wash stability compared to the VH3 binding polypeptide (SEQ ID NO:10). SEQ ID NO:13 and SEQ ID NO:15 have alkaline wash stability comparable to the alkaline wash stability positive control (SEQ ID NO:88) that binds both the Fc fragment and the Fab fragment. The VH3 binding polypeptides (SEQ ID NO:13-15) have greater alkaline wash stability than the non-Fc binding control (negative CTRL) that has no alkaline wash stability.

[0260] [Table 6]

[0261] conclusion Further novel VH3 binding polypeptides (SEQ ID NOs: 13-15) show no binding to trastuzumab Fc. Further novel VH3 binding polypeptides (SEQ ID NOs: 13-15) show improved binding to both Fab and trastuzumab compared to the VH3 binding polypeptide (SEQ ID NO: 10) tested in Example 2.

[0262] Further VH3 binding polypeptides (SEQ ID NOs: 13-15) are alkaline wash stable and retain at least 31% of their trastuzumab response in cycle 2 after 100 wash cycles with 0.5 M NaOH at 10 μl / min with a 10 minute contact time. SEQ ID NO: 14 has alkaline wash stability comparable to the VH3 binding polypeptide (SEQ ID NO: 10). SEQ ID NOs: 13 and 15 have improved alkaline wash stability compared to the VH3 binding polypeptide (SEQ ID NO: 10). SEQ ID NOs: 13 and 15 show improved binding to trastuzumab Fab compared to the alkaline wash stable positive control (SEQ ID NO: 88) which binds both the Fc and Fab fragments of trastuzumab.

[0263] It is concluded that the presence of at least amino acids at positions 9, 10, 11, 13, 14, 17, 18, 28 and 29, as shown in Table 5, confers advantageous properties to the VH3 binding polypeptides according to the invention herein, such that said VH3 binding polypeptides (SEQ ID NOs: 13-15) exhibit even further improved alkaline washing stability and exhibit binding to the Fab fragment but not to the Fc fragment of trastuzumab.

[0264] Based on the sequence information shown in Table 5 and the results presented in Example 3, it is concluded that VH3 binding polypeptides comprising glycine (G) at position 29 (SEQ ID NO: 13 and 15) exhibit surprisingly high binding affinity to Trastuzumab and the Fab fragment of Trastuzumab compared to a VH3 binding polypeptide comprising alanine (A) at position 29 (SEQ ID NO: 14) and compared to the alkaline wash stability positive control (SEQ ID NO: 88) that binds both the Fc fragment and the Fab fragment of Trastuzumab. It is further concluded that a VH3 binding polypeptide comprising alanine (A) at position 28 and glycine (G) at position 29 (SEQ ID NO: 15) has surprisingly superior alkaline wash stability characteristics compared to a VH3 binding polypeptide comprising asparagine (N) at position 28 and glycine (G) at position 29.

[0265] Example 4 Evaluation of VH3-binding polypeptide multimer variants by chromatography overview A subset of alkaline wash stable VH3 binding polypeptides in which Fc binding was abolished (Fr32 and Fr33; SEQ ID NOs: 14 and 15) were selected for further testing on the column. Further testing included multimerization of the polypeptides into tetramers and hexamers and expression of the multimers in E. coli. Primary characterization was performed on those multimeric VH3 binding polypeptides (SEQ ID NOs: 175-177) to evaluate the ligands in terms of dynamic binding capacity (DBC), pH elution and alkaline wash stability.

[0266] The effect of the A29G mutation on the tetramers of the VH3 binding polypeptides (SEQ ID NO: 175 and 176) was investigated with trastuzumab in terms of DBC and elution pH in a pH gradient. The hexamer of the VH3 binding polypeptide (SEQ ID NO: 177) was tested with trastuzumab and different target molecules in terms of DBC and elution pH in a pH gradient. In addition, the alkaline wash stability of SEQ ID NO: 177 was tested by running incubation with 0.5 M NaOH for 4 hours followed by DBC measurement. The hexamer (SEQ ID NO: 181) of the alkaline wash stable polypeptide (SEQ ID NO: 88) that binds both the Fc and Fab fragments of trastuzumab was used as a control.

[0267] Materials and Methods Plasmid and cell banks A subset of H6C-tagged multimer candidate variants was prepared according to the general formula. In particular, SEQ ID NO: 14 as a tetramer and SEQ ID NO: 15 as a tetramer and hexamer, containing sequences according to SEQ ID NO: 175-177, respectively, were ordered as DNA synthesis plasmids from ATUM Inc. (Newark, CA, USA). The plasmids were transformed into E. coli K12-017 chemically competent cells, and a research cell bank was generated according to standard procedures.

[0268] fermentation Fermentation was carried out in a continuous midstream process using the aforementioned multimer candidates.

[0269] A seed culture was prepared in a 500 mL baffled shake flask with 100 mL of Terrific Broth (TB), 100 μL of Kanamycin (50 mg / mL) and 100 μL of Research Cell Bank (RCB) of SEQ ID NO: 175-177. The seed culture was incubated at 37°C and 180 rpm agitation for 15 hours to a final OD600 value of approximately 20. The fermentation medium was prepared and sterilized in the reactor at 121°C for 20 minutes before adding additives. The reactor was inoculated at an OD600 of 0.2. The starting conditions of the culture were 37°C, pH 7.1, 300 RPM agitation, 1 L / min aeration, and DO was kept constant at 30%. When the glucose level in the reactor was below 1 g / L, glucose feed was started at a specific growth rate of 0.3 / h. Protein expression was induced using isopropyl bD-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. The culture was stopped after 25 hours and heat-treated in the reactor.

[0270] Clarification of heat-treated fermentation broth 3600 cm with a cutoff of 0.2 μM 2 The heat-treated fermentation medium was clarified using an AKTA flux system connected to a hollow fiber filter cartridge (Cytiva™, Uppsala, Sweden). The fermentation broth was concentrated three times and then dialyzed against water. The retentate was concentrated until a pressure of 2 bar was reached. Na azide was added to the harvest material to a final concentration of approximately 0.02% before storing in the cold room. The clarified feed material was loaded in the first chromatography step.

[0271] Chromatographic purification Constructs were synthesized with a hexaHis tag and purified on Ni-Sepharose 6FF (Cytiva™, Uppsala, Sweden). The clarified feed was loaded onto Ni-Sepharose 6FF resin packed in a HiScale26 / 40 column. The column was equilibrated with 50 mM phosphate, 500 mM NaCl, pH 7.5 before loading 300 mL of clarified feed. The purified proteins were eluted with a step gradient using 50 mM phosphate, 500 mM NaCl, 2 M imidazole (Merck, Darmstadt, Germany), pH 7.5. After capture with the His tag, the proteins were further purified on a Source 15Q. The eluate of the capture step was reduced by the addition of DTT to a final concentration of 50 mM for 60 min at room temperature. After reduction, the sample was desalted to 25 mM Tis pH 8 using Sephadex G50 (Cytiva™, Uppsala, Sweden) before loading onto a Source 15 Q (Cytiva™, Uppsala, Sweden). Protein was eluted from the resin with a salt gradient of 25 mM Tris pH 8, 500 mM NaCl. The final protein was clarified from E. coli HCP, aggregates and truncated forms in a polishing step. The eluted protein was concentrated to a high concentration of approximately 50 g / L using a Vivaspin 20 (Cytiva™, Uppsala, Sweden) spin column with a 5 kDa cutoff. The concentrated protein was used for coupling to the base matrix.

[0272] Coupling Coupling was performed to 60 μm and 50 μm agarose beads by using epoxy chemistry. The protein ligand was reduced in a 50 mL Falcon tube. To 20 mL of ligand solution (50 mg / mL), 169 mg NaHCO3, 21 mg Na2CO3, 175 mg NaCl and 7 mg EDTA were added. The Falcon tube was placed on a roller table for 5-10 min before adding 77 mg DTT. Reduction was continued for >45 min. The ligand solution was then desalted by using a PD10 column packed with Sephadex G-25 (Cytiva, Uppsala, Sweden). The ligand content in the desalted solution was determined by measuring the UV absorption at 276 nm. The epoxy-activated gel was washed with 3-5 gel volumes (GV) of 0.1 M phosphate, 1 mM EDTA pH 8.6 and then the ligand was coupled according to the method described in US Pat. No. 6,399,750. After immobilization, the resin was washed 3xGV with distilled water. The resin was mixed with 1GV of 0.1M phosphate, 1mM EDTA, 10% thioglycerol pH 8.6 and the tube was left on a shaking platform at room temperature overnight. The resin was then washed alternately with 3xGV of 0.1M TRIS, 0.15M NaCl pH 8.6 and 0.5M HAc, followed by 8-10xGV of distilled water. Resin samples were sent for amino acid analysis and the ligand density (LD) was calculated from the total amino acid content per mg / mL of resin.

[0273] Packing the column and taking a 1mL cube of sample To pack the same volume of resin each time, a 50% resin slurry was prepared and packed into a 1 mL cube using set vacuum. A 1 ml cube of resin sample was transferred to a Tricorn column (Cytiva™, Uppsala, Sweden) using a Pasteur pipette.

[0274] Column Packing Control The column packing was checked by injecting a 0.8 M NaCl solution through the column and calculating the resulting peak asymmetry. The column was equilibrated with 2 CV of 0.4 M NaCl at a flow rate of 1 mL / min. 10 μl of the 0.8 M NaCl solution was injected at a flow rate of 20 cm / h. The plate height and peak asymmetry coefficient were then evaluated using the AKTA™ system (Cytiva™, Uppsala, Sweden).

[0275] Preparation of trastuzumab Purified mAb solutions were diluted in PBS (Medicago, Uppsala, Sweden) and filtered through a 0.2 μm syringe filter. Concentrations were determined by measuring the UV maximum at 280 nm with an AKTA Pure25 system (Cytiva™, Uppsala, Sweden).

[0276] DBC measurement DBC measurements were performed on a Tricorn 5 / 50 column. The column was equilibrated with PBS buffer. Trastuzumab, trastuzumab Fab fragments or nanobody were loaded onto the column by the sample pump at the desired flow rate to achieve a residence time of 6 minutes (depending on the bed height of the column), typically 0.166 mL / min, until a UV signal of approximately 20% of the maximum was achieved. The column was then washed with PBS buffer at a flow rate of 1 mL / min. Proteins were eluted with elution buffer (50 mM sodium citrate pH 2.5) at a flow rate of 1 mL / min. The column was washed with 0.1 M NaOH at a flow rate of 0.166 mL / min for 15 minutes and then re-equilibrated with PBS buffer.

[0277] pH elution using a gradient pH elution measurements were performed on a Tricorn 5 / 50 column. The column was equilibrated with PBS buffer. Approximately 10 mg of trastuzumab, trastuzumab Fab fragment or nanobody was loaded onto the column by the sample pump at a desired flow rate to achieve a 6 min residence time, typically 0.166 mL / min. The column was then washed with PBS buffer at a flow rate of 1 mL / min. Protein was eluted with an elution gradient from 50 mM sodium citrate pH 6.5 to 50 mM sodium citrate pH 2.5 at a flow rate of 1 mL / min. The column was washed with 0.1 M NaOH at a flow rate of 0.166 mL / min for 15 min and then re-equilibrated with PBS buffer. The elution pH was determined as the apex of the elution peak.

[0278] Accelerated NaOH Test An accelerated NaOH test was performed by running an initial DBC run to measure resin capacity at a set residence time, then filling the column with 0.5M NaOH. When the column was completely filled, the flow was stopped and the column was incubated in NaOH for 4 hours before being equilibrated with PBS and a new DBC measurement was performed. This repetition was continued until the column had been incubated in 0.5M NaOH for a total of 24 hours.

[0279] result Evaluation of VH3-binding polypeptide tetramers by chromatography The tetramer variants (SEQ ID NO: 175 and SEQ ID NO: 176) were evaluated by DBC measurements and pH elution, and the results are summarized in Table 7.

[0280] [Table 7]

[0281] Evaluation of VH3-binding polypeptide hexamers by chromatography The hexamer variant (SEQ ID NO: 177) coupled resin was packed into a column and first tested for dynamic binding capacity (DBC) of trastuzumab, trastuzumab Fab and variable heavy fragment (VHH), the results of which are summarized in Table 8.

[0282] [Table 8]

[0283] Analysis of column stability during alkaline washing An accelerated NaOH test was performed to test the alkaline wash stability of Fr33-6 (SEQ ID NO: 177) on beads. SEQ ID NO: 181 was used as a control. In this test, DBC was measured at RT for 6 min, followed by incubation in 0.5 M NaOH for 4 h, with DBC measurements taken between each incubation. The resin was incubated for a total of 24 h. The results of the alkaline wash stability test are shown in Table 9.

[0284] [Table 9]

[0285] conclusion Column testing shows that the single A29G mutation from SEQ ID NO: 175 to SEQ ID NO: 176 reduces the elution pH and therefore indicates higher affinity. This higher affinity is also shown by Biacore in Example 3. Furthermore, the increased affinity of SEQ ID NO: 177 compared to the control results in an increased capacity for single 1:1 interactions, e.g., Fab, VHH or bispecific antibodies (Table 8). Surprisingly, these mutations could be introduced into the scaffold without significantly reducing the alkaline wash stability of the protein (Table 9).

[0286] Example 5 Selective VH interaction evaluation of VH3-binding polypeptides by Biacore overview Antibodies are composed of a heavy chain (HC) and a light chain (LC), where the HC is composed of three quantitative domains (CH1-CH3) and one variable domain (VH), and the LC is composed of one quantitative domain (CL) and one variable domain (VL). Most of the variation in the IgG1 antibody framework is in the VH and VL. Both trastuzumab and adalimumab are IgG1 antibodies with VH class 3 and VL kappa I. Both trastuzumab and adalimumab have a VH3 framework, but trastuzumab and adalimumab differ at several amino acid positions, including two positions 57 and 64 (according to the Kabat numbering system) for VH3 interactions in the protein A domain.

[0287] [Table 10]

[0288] Even though there is a high similarity between trastuzumab and adalimumab, the selective binding to the VH3 domain of the VH3-binding polypeptide or multimer thereof of the present invention is different in said antibodies. Adalimumab does not bind to the VH3-binding polypeptide multimer of the present invention. Similarly, other non-VH3 class antibodies, such as burosumab (IgG1, VH1, κI), vedolizumab (IgG1, VH1, κII) do not bind to the VH3-binding polypeptide of the present invention. On the other hand, antibodies that have more sequence differences than adalimumab compared to trastuzumab but show the same VH3 domain amino acid sequence (e.g., those listed in Table 10) show binding to the VH3-binding polypeptide or multimer thereof of the present invention. Examples of such antibodies are avelumab (IgG1, VH3, λII) and dupilumab (IgG4, VH3, κII).

[0289] In the following example, a representative polypeptide according to the present disclosure (SEQ ID NO: 177) in multimeric form is immobilized on a Biacore™ chip surface and the mAbs from Table 10 are investigated for their binding selectivity to the VH3 domain compared to other non-binding mAbs. The VH3-binding polypeptide multimer (SEQ ID NO: 177) shows no binding affinity to adalimumab, burosumab, and vedolizumab, but is predicted to bind to trastuzumab, avelumab, and dupilumab, indicating that the VH3-binding polypeptide multimer binds to the VH3 region of the mAb for trastuzumab, which is also present in avelumab and dupilumab.

[0290] Materials and Methods To assess binding to VH3, VH3-binding polypeptides are immobilized on a Biacore™ CM5 chip. Different mAbs (5000 μM) are used as analytes at a flow rate of 10 μl / min for 10 min followed by 10 min dissociation. After the run, the surface is regenerated with two injections of 10 mM glycine pH 1.5.

[0291] result The data is expected to show that the VH3 binding polypeptide multimer (SEQ ID NO: 177) binds to trastuzumab, avelumab, and dupilumab, but is not expected to bind to adalimumab, burosumab, or vedolizumab.

[0292] conclusion Based on the expected results, it is concluded that the VH3 binding polypeptide and multimers thereof according to the present invention, as exemplified by SEQ ID NO: 177, specifically bind to the VH3 domain of trastuzumab. Trastuzumab and adalimumab are highly similar except for the VH3 domain, and said VH3 binding polypeptide and multimers thereof do not bind to adalimumab, therefore it is concluded that said VH3 binding polypeptide and multimers specifically bind to the VH3 domain of trastuzumab.

[0293] VH3 class mAbs, such as avelumab and dupilumab, which have different VH3 domains than trastuzumab but the same VH3 domains as trastuzumab presented in Table 10, are expected to bind to the VH3 binding polypeptide multimer. The expected results show that the presence of residues I and E at Kabat positions 57 and 64, respectively, in the VH3 domain of adalimumab results in loss of binding to the VH3 binding polypeptide multimer (SEQ ID NO: 177). It is concluded that the VH3 binding polypeptide according to the present invention binds to the VH3 region of trastuzumab.

[0294] Example 6 Specificity testing of VH3-binding polypeptide multimers using VH1 and VH3 class targets overview Example 5 shows that certain mutations in the VH3 domain of trastuzumab are key to retain trastuzumab VH3 binding. The specificity of VH3 binding polypeptide multimers can be tested using targets with different sequences in the VH3 binding domain. Furthermore, the specificity of VH3 binding polypeptide multimers for VH3 class targets can be tested by investigating binding to both VH1 and VH3 class targets and binding to VH1 / VH3 bispecific antibodies.

[0295] Using a set of different commercially available antibodies and one bispecific antibody, we tested whether the VH3 interaction of Fr33-6 (SEQ ID NO: 177) could be used for binding and elution of antibodies from the column.

[0296] The following example shows that the VH3 binding polypeptide multimer (SEQ ID NO: 177) binds to a VH3 class target that has the same amino acids as trastuzumab at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, 82a and 82b (according to the Kabat numbering system) of the VH3 domain. Furthermore, the results show that the VH3 binding polypeptide multimer does not bind to any of the VH1-containing targets tested, but retains binding to a VH1 / VH3 bispecific antibody.

[0297] Materials and Methods A set of antibodies with different sequences within the VH3 binding domain (Table 11) and VH1 class antibodies were tested for binding to SEQ ID NO: 177 (trade names and suppliers are shown in brackets): avelumab (Bavencio, Merck KGaA), erenumab (Aimovig, Novartis), dupilumab (Dupixent, Sanofi Genzyme), denosumab (Xgeva, Amgen), bevacizumab (Avastin, Roche), adalimumab (Humira, AbbVie), emicizumab (Hemlibra, Roche), belimumab (Benlysta, GSK), guselkumab (Tremfya, Jansen), rituximab (Mabthera, Roche), pembrolizumab (Keytruda, MSD).

[0298] [Table 11]

[0299] Binding / elution (B / E) experiments were performed on a Tricorn 5 / 50 column. The column was equilibrated with PBS buffer. Approximately 10 mg of mAb diluted to 1 mg / ml in PBS was then loaded onto the column by the sample pump. The column was then washed with PBS buffer at a flow rate of 1 mL / min. Protein was eluted with 50 mM sodium citrate pH 2.5 at a flow rate of 1 mL / min. The column was washed with 0.1 M NaOH and then re-equilibrated with PBS buffer. Bound was defined as protein exiting as the elution pool and unbound if the protein entered the loading flow-through (FT).

[0300] result A set of antibodies with different sequences within the VH3 binding domain and VH1 class antibodies were tested for binding to SEQ ID NO:177.

[0301] The results are summarized in Table 12. Four of the VH3 antibodies tested, avelumab, erenumab, dupilumab, and denosumab, bound to SEQ ID NO: 177. VH3 class targets (avelumab, dupilumab, and denosumab) with the same amino acids as trastuzumab at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, 82a, and 82b (according to the Kabat numbering system) bound to the VH3-binding polypeptide multimer (SEQ ID NO: 177). Erenumab, a VH3 class target with K at position 57 and S at position 59, also bound to the VH3-binding polypeptide multimer (SEQ ID NO: 177). Two of the VH3 class antibodies, Avastin, which has a P at position 57 and an R at position 65, and Humira, which has an I at position 57 and an E at position 64, did not bind to the VH3-binding polypeptide multimer (SEQ ID NO: 177).

[0302] Hemlibra, a bispecific antibody having one VH1 domain and one VH3 domain, bound to a VH3-binding polypeptide multimer (SEQ ID NO: 177).

[0303] Of the four VH1 antibodies tested, none bound to the VH3 binding polypeptide multimer (SEQ ID NO: 177).

[0304] [Table 12]

[0305] conclusion Representative VH3 binding polypeptides according to the invention were tested. The VH3 binding polypeptide multimer (SEQ ID NO: 177) binds to VH3 class antibodies that have the same amino acids as trastuzumab at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, 82a and 82b (according to the Kabat numbering system) of the VH3 domain. SEQ ID NO: 177 was shown not to bind to VH1 class antibodies, but instead binds to bispecific antibodies with at least one VH3 domain. It is concluded that the VH3 binding polypeptides according to the invention bind specifically to the VH3 region present in trastuzumab.

[0306] It is further concluded that the VH3 interaction of the VH3 binding polypeptide or multimer thereof according to the invention exemplified by SEQ ID NO: 177 can be used for antibody purification even when the Fc interaction is lost. This interaction works for some VH3 class antibodies, but not all of them. Furthermore, none of the VH1 class antibodies bound to the resin containing SEQ ID NO: 177. Interestingly, bispecific antibodies with one binding arm can be purified in B / E mode, indicating that only one interaction is sufficient to be used as a purification tool.

[0307] Example 7 Separation of VH1 / VH3 bispecific antibodies from VH3 / VH3 homodimers using VH3-binding polypeptide multimers overview This example describes the separation of VH1 / VH1 and VH3 / VH3 homodimers from asymmetric bispecific antibody heterodimers consisting of paired heavy chains of the VH1 / VH3 class. For the production of asymmetric VH1 / VH3 bispecific antibodies, the two heavy chains must be correctly paired. The mis-paired heavy chains are VH1 / VH1 or VH3 / VH3 homodimers.

[0308] As shown in Example 6 and Table 12, VH1 / VH1 homodimers do not bind to SEQ ID NO: 177 and therefore pass through the resin coupled to the alkaline wash stable VH3 binding polypeptide hexamer (SEQ ID NO: 177) without binding. In the following example, it is demonstrated that VH3 / VH3 homodimers show stronger binding to the resin coupled to the alkaline wash stable VH3 binding polypeptide hexamer (SEQ ID NO: 177). Without being bound by theory, it is expected that this is due to an avidity effect created by the presence of two VH3 targets in the homodimer. Therefore, VH3 / VH3 homodimers can be eluted in the gradient at a lower pH than the asymmetric VH1 / VH3 bispecific antibody.

[0309] It is concluded that the resin coupled to the alkaline wash stable VH3 binding polypeptide or multimer thereof, such as the hexamer (SEQ ID NO: 177), has advantageous properties that allow simultaneous chromatographic separation of VH1 / VH1 and VH3 / VH3 homodimers from VH1 / VH3 heterodimers. By utilizing this effect of the resin, it is possible to separate mismatched homodimers from correctly paired heterodimers. Thus, the resin comprising the alkaline wash stable VH3 binding polypeptide or multimer thereof according to the present invention allows the purification of asymmetric VH1 / VH3 bispecific antibodies or multimers thereof in an excellent manner using a single chromatographic step.

[0310] Separation of homodimers and heterodimers in emicizumab (Hemlibra®) feed Materials and Methods Emicizumab was ordered from Thermo Fisher (GeneArt, Regensburg, Germany) according to the published sequence (KEGG Drug Database). The bispecific antibody was produced in Expi293 cells, concentrated 10x and delivered in sterile filtered cell culture medium. The target protein concentration was approximately 1 g / L as measured by SDS-PAGE. The cell culture medium was loaded onto either a control column containing a resin coupled to SEQ ID NO: 181 (as an Fc and VH3 binding control) or a column containing a resin coupled to SEQ ID NO: 177, which binds only via VH3 interaction according to the invention. The protein of SEQ ID NO: 177 was coupled to two agarose-based matrices of 60 and 50 μm to see if the bead size could affect the resolution. Approximately 5 mL of feed was loaded onto a 1 mL column packed in a Tricorn 5 / 50 and eluted with the following buffers: equilibration buffer, a gradient from pH 6.5 to 2.5 using 20 mM sodium phosphate in 150 mM sodium chloride, pH 7.2, a gradient from 50 mM sodium citrate pH 6.5 to 50 mM sodium citrate pH 2.5, followed by a 0.5 M NaOH wash. Once the cycle was completed, the column was regenerated again with equilibration buffer. To separate the homodimers and heterodimers, LC / MS was used to measure the mass of protein in each elution fraction.

[0311] result The emicizumab feed contains VH1 / VH3 bispecific antibodies and also VH3 / VH3 mismatched homodimers. Only one elution peak can be detected using a control column containing a resin coupled to SEQ ID NO: 181, a hexamer of an alkaline wash-stable polypeptide (SEQ ID NO: 88) that binds both the Fc fragment and the Fab fragment of trastuzumab (Figure 4A). Thus, the control column containing a resin coupled to SEQ ID NO: 181 does not allow the VH1 / VH3 bispecific antibodies and VH3 / VH3 mismatched homodimers in the emicizumab feed to be separated from each other. In contrast, two elution peaks can be detected using a column containing a resin according to the invention coupled to SEQ ID NO: 177, a hexamer of an alkaline wash-stable VH3 binding polypeptide (SEQ ID NO: 15) that does not bind the Fc fragment of trastuzumab (Figures 4B and 4C). Thus, the VH1 / VH3 bispecific antibody and the VH3 / VH3 mispaired homodimer in the emicizumab feed can be easily separated from each other on a column containing a resin according to the invention coupled to SEQ ID NO: 177. Resolution was highest with the smaller 50 μm bead size (FIG. 4C). Eluate fractions from the 60 μm beads were analyzed by LC / MS. The homodimer is theoretically 485 Da larger than the heterodimer based on the amino acid sequence and could be detected in fractions 7 and 8 (Table 13). The results show that the homodimer elutes at a slightly lower pH due to the dual interaction.

[0312] [Table 13]

[0313] Mismatched VH3 / VH3 homodimers bind more strongly to SEQ ID NO: 177 resin than VH1 / VH3-binding heterodimers of bispecific antibodies. This is expected to be due to the presence of multiple binding sites and avidity effects. Thus, VH3 / VH3 homodimers are eluted at lower pH on columns containing resins coupled to SEQ ID NO: 177 according to the present invention. Thus, in the chromatograms of Figures 4B and 4C, there is a distinctive peak eluting at about pH 2.9, corresponding to the elution of VH3 / VH3 homodimers, and at higher pH, corresponding to the VH1 / VH3 bispecific antibody.

[0314] conclusion It is concluded that separation of mismatched homodimers, as opposed to heterodimers, can be achieved by either the VH3 binding polypeptide according to the invention or its multimers, as shown here for Fr33-6 (SEQ ID NO: 177). This separation is based on binding to antibody VH3 interactions. If an antibody has two VH3 domains that are mismatched and therefore can interact with the polypeptide of the invention, it will bind more tightly to the column and elute at a slightly more acidic pH. This separation can be achieved due to the elimination of the Fc interaction of Protein A.

[0315] It is concluded that a resin composed of an alkaline wash stable VH3 binding polypeptide or a multimer thereof, such as a hexamer (SEQ ID NO: 177), has advantageous properties that allow chromatographic separation of mismatched VH1 / VH1 and VH3 / VH3 homodimers from a VH1 / VH3 bispecific antibody. Separation of VH1 / VH1 and VH3 / VH3 homodimers from a VH1 / VH3 bispecific antibody is made possible by the advantageous characteristics of an alkaline wash stable VH3 binding polypeptide or a multimer thereof. As shown herein, the alkaline wash stable VH3 binding polypeptide hexamer (SEQ ID NO: 177) as well as the alkaline wash stable VH3 binding polypeptide that does not bind to the Fc fragment of trastuzumab (SEQ ID NO: 15) do not bind to the Fc fragment and VH1 domain of the antibody, but show high binding affinity to the VH3 domain of the antibody. The alkaline wash stability properties of said polypeptides and multimers thereof allow the use of said VH3 binding polypeptides and / or multimers in the chromatographic separation of VH1 / VH1 and VH3 / VH3 homodimers from VH1 / VH3 bispecific antibodies. Moreover, said properties allow the repeated use of the resins containing said polypeptides and / or multimers, with intermediate wash steps, whilst retaining significantly their binding properties.

[0316] Utilizing this superior effect of the resin composed of the alkaline wash stable VH3 binding polypeptide or multimer thereof according to the present invention revolutionizes the production of bispecific antibodies, making them more affordable to produce and thus facilitating the production of therapeutics comprising bispecific antibodies. The simultaneous purification of correctly paired VH1 / VH3 bispecific antibodies from VH1 / VH1 and VH3 / VH3 homodimers is advantageous for the production of highly pure VH1 / VH3 bispecific antibody therapeutics.

[0317] Example 8 Evaluation of VH3 binding polypeptides in different scaffolds To evaluate the effect of mutations at positions 9, 10, 11, 13, 14, 17, 18, 28 and 29 in different scaffolds, SEQ ID NO:89 was modified at nine positions with amino acid residues according to Tables 1, 3 and 5. The resulting VH3 binding polypeptides are listed as SEQ ID NOs:42-56.

[0318] VH3 binding polypeptides (SEQ ID NOs: 42-45) are produced essentially as described in Example 1 under the section entitled "Generation of His-purified candidate polypeptides." Evaluation and analysis are performed essentially as described in Example 1 under the sections entitled "Biacore analysis of binding affinity to Fc and Fab fragments of trastuzumab" and "Biacore analysis of alkaline wash stability."

[0319] The VH3 binding polypeptides (SEQ ID NO: 42-45) are evaluated along with a control (SEQ ID NO: 89) that binds to both the Fc and Fab fragments of trastuzumab, a non-Fc binding control (SEQ ID NO: 180) and a non-Fc binding control with no alkaline wash stability (negative CTRL).

[0320] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of Fab interactions (tested by injecting high concentrations of Fab fragments generated from trastuzumab) (3) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) Rate the following.

[0321] VH3 binding polypeptides (SEQ ID NOs: 46-53) are produced essentially as described in Example 2 under the section entitled "Generation of His-purified candidate polypeptides." Evaluation and analysis are performed essentially as described in Example 2 under the sections entitled "Biacore analysis of binding affinity to Fc fragment of trastuzumab and trastuzumab" and "Biacore analysis of alkaline wash stability."

[0322] The VH3 binding polypeptides (SEQ ID NOs: 46-53) are evaluated along with a control (SEQ ID NO: 89) that binds to both the Fc and Fab fragments of trastuzumab and a non-Fc binding control with no alkaline wash stability (negative CTRL). The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of trastuzumab interaction (tested by injecting high concentrations of trastuzumab) (3) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) Rate the following.

[0323] VH3 binding polypeptides (SEQ ID NOs:54-56) are generated essentially as described in Example 3 under the section entitled "Generation of His-purified candidate polypeptides." Evaluation and analysis are performed essentially as described in Example 3 under the sections entitled "Biacore analysis of binding affinity to Fc and Fab fragments of trastuzumab" and "Biacore analysis of alkaline wash stability."

[0324] The VH3 binding polypeptides (SEQ ID NOs: 54-56) are evaluated along with a control (SEQ ID NO: 89) that binds to both the Fc and Fab fragments of trastuzumab and a non-Fc binding control with no alkaline wash stability (negative CTRL).

[0325] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of Fab interactions (tested by injecting high concentrations of Fab fragments generated from trastuzumab) (3) Affinity assessment of trastuzumab interaction (tested by injecting high concentrations of trastuzumab) (4) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) Rate the following.

[0326] The VH3 binding polypeptides (SEQ ID NOs: 42-56) are predicted to be alkaline wash stable or exhibit improved alkaline wash stability properties, exhibit VH3 binding affinity, and exhibit significantly reduced or eliminated affinity for the Fc region of trastuzumab compared to SEQ ID NO: 89.

[0327] Example 9 Evaluation of additional VH3 binding polypeptides in additional scaffolds Position X9X in further scaffold 10 X 11 X 13 X 14 X 17 X 18 X 28 X 29 To evaluate the effect of the amino acids "AYRLAARAG", SpA protein native domains A (SEQ ID NO: 92), B (SEQ ID NO: 91), C (SEQ ID NO: 90), D (SEQ ID NO: 93) and E (SEQ ID NO: 94) are modified with amino acid residues having the same amino acids at positions 9, 10, 11, 13, 14, 17, 18, 28 and 29 as shown in Table 5 for the VH3 binding polypeptide Fr33 (SEQ ID NO: 15). The VH3 binding polypeptide (AFr33) obtained in the background of SEQ ID NO: 92 is listed as SEQ ID NO: 85, the VH3 binding polypeptide (BFr33) obtained in the background of SEQ ID NO: 91 is listed as SEQ ID NO: 84, the VH3 binding polypeptide (CFr33) obtained in the background of SEQ ID NO: 90 is listed as SEQ ID NO: 83, the VH3 binding polypeptide (DFr33) obtained in the background of SEQ ID NO: 93 is listed as SEQ ID NO: 86 and the VH3 binding polypeptide (EFr33) obtained in the background of SEQ ID NO: 94 is listed as SEQ ID NO: 87.

[0328] VH3 binding polypeptides (SEQ ID NOs:83-87) are produced essentially as described in Example 3 under the section entitled "Generation of His-purified candidate polypeptides." Evaluation and analysis are performed essentially as described in Example 3 under the sections entitled "Biacore analysis of binding affinity to Fc and Fab fragments of trastuzumab" and "Biacore analysis of alkaline wash stability."

[0329] VH3 binding polypeptides (SEQ ID NOs: 83-87) are evaluated pairwise against their corresponding scaffold polypeptides (SEQ ID NOs: 90-94) that bind both the Fc and Fab fragments of trastuzumab, along with a non-Fc binding control (negative CTRL) that has no alkaline wash stability.

[0330] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of Fab interactions (tested by injecting high concentrations of Fab fragments generated from trastuzumab) (3) Affinity assessment of trastuzumab interaction (tested by injecting high concentrations of trastuzumab) (4) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) Rate the following.

[0331] The VH3 binding polypeptides (SEQ ID NOs: 83-87) are predicted to be alkaline wash stable or exhibit improved alkaline wash stability properties, exhibit VH3 binding affinity, and exhibit significantly reduced or eliminated affinity for the Fc region of trastuzumab compared to their corresponding scaffold polypeptides (SEQ ID NOs: 90-94).

[0332] Example 10 Evaluation of additional VH3 binding polypeptides in two different scaffolds To evaluate the effect of mutations at positions 13 and 14 in two different scaffolds, alkaline wash stable VH3 binding polypeptides (Fr33) (SEQ ID NO: 15 and SEQ ID NO: 56) are modified at two positions with amino acid residues according to Table 14. The VH3 binding polypeptides obtained in the background of SEQ ID NO: 15 are listed as SEQ ID NO: 16 to 40, and the VH3 binding polypeptides obtained in the background of SEQ ID NO: 56 are listed as SEQ ID NO: 57 to 81. Furthermore, the effect of a glutamine (Q) to serine (S) mutation at position 26 is evaluated in alkaline wash stable VH3 binding polypeptides (Fr33) (SEQ ID NO: 15 and SEQ ID NO: 56) in two different scaffolds. The VH3 binding polypeptide (VH3K2) obtained in the background of SEQ ID NO: 15 is listed as SEQ ID NO: 41, and the VH3 binding polypeptide (VH3K2) obtained in the background of SEQ ID NO: 56 is listed as SEQ ID NO: 82.

[0333] [Table 14]

[0334] VH3 binding polypeptides (SEQ ID NOs: 16-41 and SEQ ID NOs: 57-82) are produced essentially as described in Example 3 under the section entitled "Generation of His-purified candidate polypeptides." Evaluation and analysis are performed essentially as described in Example 3 under the sections entitled "Biacore analysis of binding affinity to Fc and Fab fragments of trastuzumab" and "Biacore analysis of alkaline wash stability."

[0335] The VH3 binding polypeptides (SEQ ID NOs: 16-41) are evaluated along with a control (SEQ ID NO: 88) that binds to both the Fc and Fab fragments of trastuzumab and a non-Fc binding control with no alkaline wash stability (negative CTRL).

[0336] The VH3 binding polypeptides (SEQ ID NOs: 57-82) are evaluated along with a control (SEQ ID NO: 89) that binds to both the Fc and Fab fragments of trastuzumab and a non-Fc binding control with no alkaline wash stability (negative CTRL).

[0337] The following aspects: (1) Affinity assessment of Fc interactions (tested by injecting high concentrations of Fc fragments generated from trastuzumab) (2) Affinity assessment of Fab interactions (tested by injecting high concentrations of Fab fragments generated from trastuzumab) (3) Affinity assessment of trastuzumab interaction (tested by injecting high concentrations of trastuzumab) (4) Alkaline wash stability (tested by loss of trastuzumab binding after increasing numbers of treatments with 0.5 M NaOH) Rate the following.

[0338] The VH3 binding polypeptides (SEQ ID NOs: 16-41) are predicted to be alkaline wash stable or exhibit improved alkaline wash stability properties, exhibit VH3 binding affinity, and exhibit significantly reduced or eliminated affinity for the Fc region of trastuzumab compared to SEQ ID NO: 88.

[0339] The VH3 binding polypeptides (SEQ ID NOs:57-82) are predicted to be alkaline wash stable or exhibit improved alkaline wash stability properties, exhibit VH3 binding affinity, and exhibit significantly reduced or eliminated affinity for the Fc region of trastuzumab compared to SEQ ID NO:89.

[0340] Example 11 Biacore evaluation of IgG binding polypeptides following knockout mutations for Fc interactions Biacore analysis of binding affinity of trastuzumab to Fc and Fab fragments To evaluate binding to Fc (sequence number 184) and VH3 / Fab (sequence numbers 182 and 183), the IgG-binding polypeptide before mutation (group 1; G1) and the polypeptide after Fc interaction knockout mutation (groups 2 to 9) were immobilized on a Biacore CM5 chip, and the Fc fragment of trastuzumab (500 nM) and the Fab fragment of trastuzumab (500 nM) were used as analytes.

[0341] Each Fc knockout has the following mutations (at positions corresponding to the following positions in SEQ ID NO:89): Group 2 (G2): 9A, 10Y, 11R, 13L, 14A, 17A, 18R, 28A, 29G Group 3 (G3): 9A, 10Y, 11R, 13L, 14A, 17A, 18R, 26S, 28A, 29G Group 4 (G4): 9A, 10Y, 11E, 13R, 14A, 17A, 18R, 28A, 29G Group 5 (G5): 9A, 10Y, 11E, 13R, 14A, 17A, 18L, 28A, 29G Group 6 (G6): 9A, 10Q, 11R, 13R, 14A, 17L, 18R, 28A, 29G Group 7 (G7): 9A, 10Y, 11E, 13L, 14R, 17A, 18R, 28A, 29G Group 8 (G8): 9A, 10Y, 11E, 13A, 14R, 17A, 18R, 28A, 29G Group 9 (G9): 9Y, 10Q, 11R, 13L, 14A, 17A, 18L, 28A, 29G

[0342] The sequences contained in each group are set forth below in Table 15 and are referred to as sequence numbers (SIDs).

[0343] [Table 15]

[0344] Thus, each row in Table 15 refers to the same base sequence, the sequence shown in the column marked G1. The following columns indicate which sequences belong to each group for that particular base sequence.

[0345] The constructs were expressed and purified as disclosed above.

[0346] Biacore binding analysis: Materials and equipment used were as follows: CM5 sensor chip (Cytiva™), Biacore™ NHS Coupling Kit (Cytiva™), polypeptides (SEQ ID NO:89 to SEQ ID NO:94 and SEQ ID NO:185 to SEQ ID NO:239); Biacore™ 8K+ (Cytiva™); Fab and Fc fragments (prepared from in-house trastuzumab as disclosed above).

[0347] Immobilization was performed by coupling of the polypeptide variants in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1) using standard methods in the Biacore software. The polypeptide variants used for immobilization were diluted in a concentration range of 10-50 μg / ml in acetate buffer (Cytiva™) at a pH value ≧1 unit below the pI of the polypeptide. The immobilization levels differed somewhat between the different polypeptide variants (approximately 700-2000 Ru, see Table 16).

[0348] In each run, the polypeptide was immobilized in FC2. Multiple chips were used until all candidates were tested. Biacore™ methods for binding analysis: Running buffer: PBS-P+ Flow rate: 10μl / min Sample injection: 600s / 10min on both flow cells (FC1 and FC2) Dissociation time: 600s / 10min Regeneration: 10 mM glycine-HCl pH 1.5, 30 μl / min, 2 × 30 s

[0349] Injection of the analyte Fab and Fc fragments of trastuzumab at a concentration of 500 nM in running buffer.

[0350] All sensorgrams were generated by control subtraction and responses were generated as the difference between the pre-injection baseline and the signal just prior to the end of the infusion.

[0351] result Biacore analysis of binding of trastuzumab to Fc and Fab fragments The response levels of trastuzumab Fc and Fab interactions of the polypeptide variants are summarized in Table 16 (levels rounded to the nearest multiple of 50).

[0352] [Table 16A]

[0353] [Table 16B]

[0354] In group 1, the polypeptide sequences of SEQ ID NO: 90 to SEQ ID NO: 94 (non-mutated wt domain) and SEQ ID NO: 191 show binding to the Fc fragment as well as the Fab (VH3) of trastuzumab. The polypeptide sequences of SEQ ID NO: 89, SEQ ID NO: 185 to SEQ ID NO: 190, and SEQ ID NO: 192 and SEQ ID NO: 193 do not show binding to the Fab (VH3) fragment of trastuzumab.

[0355] The data show that IgG-binding polypeptide variants that have undergone Fc knockout mutations according to group 2 (SEQ ID NOs: 194-208) have abolished or significantly reduced binding affinity to the Fc fragment of trastuzumab compared to their corresponding sequences in the original / parent polypeptide (group 1).

[0356] Different mutations to the knockout Fc interaction may affect different VH3 interactions (Fab interactions) on different IgG polypeptides in terms of stronger or weaker VH3 / Fab interactions, see Table 16.

[0357] Interestingly, it can be observed that in some cases, for example SEQ ID NO:89, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:192, mutations in both groups 2 and 3 (see SEQ ID NO:199 to SEQ ID NO:202 and SEQ ID NO:207, SEQ ID NO:212 to SEQ ID NO:215 and SEQ ID NO:220) provided Fab binding activity that was not present in the same domains in group 1, whilst Fc binding activity was lost.

[0358] SEQ ID NO:188-SEQ ID NO:190 have no Fab binding in Group 1, and the mutations according to the present disclosure did not provide Fab binding, however, Fc binding was effectively knocked out by the mutations in the test groups (Groups 2 and 3).

[0359] Thus, the mutations according to the present disclosure are shown to improve or retain Fab binding activity already present in various Protein A domains or Protein A domain variants, while also knocking out previously present Fc binding activity. In some cases, Fab binding activity can be further introduced if not previously present in the domain, but this was not the case for all domains that originally lacked Fab binding activity. However, all domains that originally had Fab / VH3 binding activity showed retained or improved Fab / VH3 binding activity and essentially abolished Fc binding activity.

[0360] Thus, the claimed polypeptides are shown to be VH3 binding polypeptides with weak or no Fc binding activity.

[0361] Table of embodiments 1. A VH3-binding polypeptide or any domain thereof derived from Staphylococcus aureus protein A (SpA), which has binding affinity to the VH3 region of trastuzumab and has lower binding affinity to the Fc region of trastuzumab compared to the binding affinity of SEQ ID NO: 88 or SEQ ID NO: 89 to the same Fc region, and is alkaline wash stable. 2. A VH3 binding polypeptide comprising sequence A, said sequence A being: i) X8X9X 10 X 11 AX 13 X 14 X 15 X 16 X 17 X 18 X 19 PNLX 23 X 24 X 25 X 26 R X 28 X 29 FIQSLX 35 X 36 (SEQ ID NO:95) (Independently of each other, X8 is selected from E, D and A; X 15 is selected from E and Q; X 16 is selected from I and V; X 19 is selected from L and M; X 23 is selected from N and T; X 24 is selected from A and E, for example, E; X 25 is selected from D and E, for example E; X 35 is selected from K, R and H; X 36 is selected from D and H); ii) an amino acid sequence having at least 83% identity to the sequence defined under i); iii) an amino acid sequence having at least 70% identity to any sequence selected from the group consisting of residues 8 to 36 in SEQ ID NO: 88, residues 8 to 36 in SEQ ID NO: 89, residues 8 to 36 in SEQ ID NO: 90, residues 8 to 36 in SEQ ID NO: 91, residues 8 to 36 in SEQ ID NO: 92, residues 11 to 39 in SEQ ID NO: 93, and residues 1 to 29 in SEQ ID NO: 94. and consisting of amino acids selected from i), ii), and iii) defined as follows: Further, in each of i), ii) and iii), independently of one another, X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11 is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G; VH3 binding polypeptides. 3. The VH3-binding polypeptide according to item 2, wherein the VH3-binding polypeptide has binding affinity to the VH3 region of trastuzumab and has a lower binding affinity to the Fc region of trastuzumab than SEQ ID NO: 88 or SEQ ID NO: 89. 4. The VH3-binding polypeptide according to any one of items 1 to 3, wherein the trastuzumab is defined by the heavy chain amino acid sequence according to SEQ ID NO: 183 and the light chain amino acid sequence according to SEQ ID NO: 184. 5. A VH3-binding polypeptide according to any one of items 2 to 4, wherein X9 is selected from Q and Y. 6. A VH3-binding polypeptide according to any one of items 2 to 4, wherein X9 is selected from Q and A. 7. A VH3-binding polypeptide according to any one of items 2 to 4, wherein X9 is selected from A and Y. 8. A VH3 binding polypeptide according to any one of items 2 to 4, 6 and 7, wherein X9 is A. 9. A VH3-binding polypeptide according to any one of items 2 to 4, 5 and 7, wherein X9 is Y. 10. A VH3 binding polypeptide according to any one of items 2 to 6, wherein X9 is Q. 11. X 10 is Q. 12. X 10 is Y. 13. X 11 is selected from T and E. 14. X 11 is selected from T and R. 15. X 11 is selected from R and E. 16. X 11 is T. 17. X 11 is E. 18. X 11 16. The VH3 binding polypeptide according to any one of items 2 to 12, 14 and 15, wherein 19. X 13are selected from L, E, R and A; or from L, E, R and Q; or from L, E, A and Q; or from L, R, A and Q; in particular from L, E, R and A; or from L, E, R and Q; or from L, E, A and Q; or from L, R, A and Q. 20. X 13 19. The VH3 binding polypeptide according to any one of items 2 to 18, wherein is selected from L, E and R; or from L, E and A; or from L, E and Q; or from L, R and A; or from L, R and Q; or from L, A and Q; or from E, R and A; or from E, R and Q; or from E, A and Q; or from R, A and Q; in particular selected from L, E and R; or from L, E and A; or from L, E and Q; or from L, R and A; or from L, R and Q. 21. X 13 19. The VH3 binding polypeptide according to any one of items 2 to 18, wherein is selected from L and E; or from L and R; or from L and A; or from L and Q; or from E and R; or from E and A; or from E and Q; or from R and A; or from R and Q; or from A and Q; in particular from L and E; or from L and R; or from L and A; or from L and Q. 22. X 13 is L. 23. X 13 22. The VH3 binding polypeptide according to any one of items 2 to 21, wherein 24. X 13 is E. 25. X 13 22. The VH3 binding polypeptide according to any one of items 2 to 21, wherein 26. X 13 22. The VH3 binding polypeptide according to any one of items 2 to 21, wherein 27. X 1427. The VH3 binding polypeptide according to any one of items 2 to 26, wherein is selected from A, W, E, L and Q; or from A, W, E, L and R; or from A, W, E, Q and R; or from A, W, L, Q and R; or from A, E, L, Q and R; or from W, E, L, Q and R. 28. X 14 27. The VH3 binding polypeptide according to any one of items 2 to 26, wherein is selected from A, W, E and L; or from A, W, E and Q; or from A, W, E and R; or from A, W, L and Q; or from A, W, L and R; or from A, W, Q and R; or from A, E, L and Q; or from A, E, L and R; or from A, E, Q and R; or from A, L, Q and R; or from W, E, L and Q; or from W, E, L and R; or from W, E, Q and R; or from W, L, Q and R; or from E, L, Q and R. 29. X 14 27. The VH3 binding polypeptide according to any one of items 2 to 26, wherein is selected from A, W and E; or from A, W and L; or from A, W and Q; or from A, W and R; or from A, E and L; or from A, E and Q; or from A, E and R; or from A, L and Q; or from A, L and R; or from A, Q and R; or from W, E and L; or from W, E and Q; or from W, E and R; or from W, L and Q; or from W, L and R; or from W, Q and R; or from E, L and Q; or from E, L and R; or from E, Q and R; or from L, Q and R. 30. X 14 27. The VH3 binding polypeptide according to any one of items 2 to 26, wherein is selected from A and W; or from A and E; or from A and L; or from A and Q; or from A and R; or from W and E; or from W and L; or from W and Q; or from W and R; or from E and L; or from E and Q; or from E and R; or from L and Q; or from L and R; or from Q and R. 31. X 14 31. The VH3 binding polypeptide according to any one of items 2 to 30, wherein 32. X 14is W. 33. X 14 is E. 34. X 14 is L. 35. X 14 is Q. 36. X 14 is R. 37. X 17 37. The VH3 binding polypeptide according to any one of items 2 to 36, wherein is selected from A and H. 38. X 17 is selected from A and L. 39. X 17 37. The VH3 binding polypeptide according to any one of items 2 to 36, wherein is selected from H and L. 40. X 17 39. The VH3 binding polypeptide according to any one of items 2 to 38, wherein 41. X 17 40. The VH3 binding polypeptide according to any one of items 2 to 37 and 39, wherein 42. X 17 is L. 43. X 18 is selected from R and L. 44. X 18 is selected from R and H. 45. X 18 is selected from H and L. 46. ​​X 18is R. 47. X 18 is L. 48. X 18 46. ​​The VH3-binding polypeptide according to any one of items 2 to 42, 44 and 45, wherein 49. X 26 is Q. 50. X 26 is S. 51. X 28 is N. 52. X 28 51. The VH3 binding polypeptide according to any one of items 2 to 50, wherein 53. X 29 is selected from A and G. 54. X 29 54. The VH3 binding polypeptide according to any one of items 2 to 53, wherein 55. X 29 is G. 56. X 14 is A and X 10 is Y or X9X 10 56. The VH3-binding polypeptide according to any one of items 2 to 55, wherein is selected from AY. 57. X9X 10 X 11 is selected from the group consisting of QQT, QYT, YQT, AQE, AQR and AYR; for example, the group consisting of QQT, QYT, AQE, AQR and AYR; for example, the group consisting of AQE, AQR and AYR; for example, the group consisting of AQR and AYR, for example, X9X 10 X 1157. The VH3-binding polypeptide according to any one of items 2 to 56, wherein said VH3-binding polypeptide is AYR. 58. X 17 X 18 is selected from the group consisting of AR, HL and LH; for example, the group consisting of AR and HL, for example, X 17 X 18 58. The VH3 binding polypeptide according to any one of items 2 to 57, wherein 59. X 24 X 25 59. The VH3 binding polypeptide according to any one of items 2 to 58, wherein 60. X 28 X 29 is selected from the group consisting of NA, NG, AA and AG; such as the group consisting of NA, NG and AG or the group consisting of NG, AA and AS; such as the group consisting of NG and AG or the group consisting of AG and NA or the group consisting of AG and AA. 61. X 28 X 29 is NA or AG or AA or NG. 62. Independently of each other, X9X 10 X 11 is selected from the group consisting of AQE, AQR and AYR; 13 X 14 is selected from the group consisting of LA, LW, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, and RR; For example, independently of each other, X9X 10 X 11 is selected from the group consisting of AQE, AQR and AYR; 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, and RR; For example, independently of each other, X9X 10 X11 is selected from the group consisting of AQR and AYR; 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ and RR; for example, XX 10 X 11 is AYR, and X 13 X 14 62. The VH3 binding polypeptide according to any one of items 2 to 61, wherein is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ and RR. 63. X9X 10 X 11 X 13 X 14 is selected from the group consisting of QQTLA, QYTLA, YQTLA, AQELA, AQRLA, AYRLA and AYRLW; for example, the group consisting of QQTLA, QYTLA, AQELA, AQRLA, AYRLA and AYRLW; for example, the group consisting of AQELA, AQRLA, AYRLA and AYRLW; for example, the group consisting of AQRLA and AYRLA; for example, X9X 10 X 11 X 13 X 14 63. The VH3 binding polypeptide according to any one of items 2 to 62, wherein is AYRLA. 64. Independently of each other, X9X 10 X 11 is selected from the group consisting of AQE, AQR and AYR; 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR, and LW; 17 X 18 is selected from LH and AR; For example, independently of each other, X9X 10 X 11is selected from the group consisting of AQR and AYR; 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR, and LW; 17 X 18 is selected from LH and AR; For example, independently of each other, X9X 10 X 11 is AYR;X 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR, and LW; 17 X 18 is selected from LH and AR; For example, independently of each other, X9X 10 X 11 is AYR;X 13 X 14 is selected from the group consisting of LA, AA, AE, AL, AQ, AR, EA, EE, EL, EQ, ER, LA, LE, LL, LQ, LR, QA, QE, QL, QQ, QR, RA, RE, RL, RQ, RR, and LW; 17 X 18 64. The VH3 binding polypeptide according to any one of items 2 to 63, wherein 65. X9X 10 X 11 X 13 X 14 X 17 X 1865. The VH3 binding polypeptide according to any one of items 2 to 64, wherein said VH3 binding polypeptide is selected from the group consisting of QQTLALH, QYTLALH, YQTLALH, QQTLAAR, AQELALH, AQRLALH, AYRLALH, AYRLWLH, AYRLWAR, AYRLAAR, AYRLAHL and AYRLWHL; such as the group consisting of QQTLALH, QYTLALH, QQTLAAR, AQELALH, AQRLALH, AYRLWLH, AYRLWAR, AYRLAAR, AYRLAHL and AYRLWHL; such as the group consisting of AQELALH, AQRLALH, AYRLALH, AYRLWAR, AYRLAAR and AYRLAHL; such as the group consisting of AQRLALH, AYRLALH and AYRLAAR; such as the group consisting of AQRLALH and AYRLAAR. 66. X9X 10 X 11 X 13 X 14 X 17 X 18 66. The VH3 binding polypeptide according to any one of items 2 to 65, wherein 67. X 24 X 25 X 28 X 29 is selected from the group consisting of EENA, EENG, EEAA and EEAG; for example, the group consisting of EENA, EENG and EEAG; for example, the group consisting of EENG and EEAG; for example, X 24 X 25 X 28 X 29 66. The VH3 binding polypeptide according to any one of items 2 to 65, wherein said VH3 binding polypeptide is EEAG. 68. X 26 X 28 X 29 is selected from the group consisting of QNG, QAA, QAG, QNA and SAG; for example, the group consisting of QNG, QAG, QNA and SAG; for example, the group consisting of QNG, QAG and SAG and / or the group consisting of QNG, QAG, QNA or the group consisting of QNG, QNA and SAG; for example, the group consisting of QAG and SAG; for example, X 26 X 28 X 2968. The VH3 binding polypeptide according to any one of items 2 to 67, wherein is QAG or SAG. 69. X 26 X 28 X 29 is selected from the group consisting of QAG and SAG; for example, X 26 X 28 X 29 69. The VH3 binding polypeptide according to any one of items 2 to 68, wherein 70. The VH3-binding polypeptide according to any one of items 2 to 69, wherein sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, such as the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, such as the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56. 71. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 41, for example, the group consisting of SEQ ID NOs: 1 to 40, for example, the group consisting of SEQ ID NOs: 1 to 15. 72. The sequence i) is a group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: A VH3-binding polypeptide according to item 70, which corresponds to a sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54, and 56 to 81; for example, a sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 13, 15, 54, and 56. 73. A VH3-binding polypeptide according to item 70, wherein the sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82; for example, the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or the group consisting of SEQ ID NOs: 15, 41, 56 and 82. 74. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56. 75. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15. 76. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the group consisting of SEQ ID NOs: 15 to 41; for example the group consisting of SEQ ID NOs: 15 to 40 or the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 15 and 41. 77. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 14 and 15. 78. A VH3-binding polypeptide according to item 70, wherein the sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 42, 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56. 79. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the group consisting of SEQ ID NOs: 56 to 82; for example the group consisting of SEQ ID NOs: 56 to 81 or the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 56 and 82. 80. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 55 and 56. 81. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the sequence from positions 8 to 36 in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. 82. A VH3-binding polypeptide according to item 70, wherein sequence i) corresponds to the sequence from positions 8 to 36 in SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. 83. A VH3-binding polypeptide according to any one of items 2 to 4, having a sequence iii) selected from the group consisting of: a sequence corresponding to residues 8 to 36 in SEQ ID NO: 83; a sequence corresponding to residues 8 to 36 in SEQ ID NO: 84; a sequence corresponding to residues 8 to 36 in SEQ ID NO: 85; a sequence corresponding to residues 11 to 39 in SEQ ID NO: 86; and a sequence corresponding to residues 1 to 29 in SEQ ID NO: 87. 84. A VH3-binding polypeptide according to any one of the preceding claims, wherein the SpA-derived polypeptide or sequence A forms part of a three-helix bundle protein domain. 85. The VH3-binding polypeptide according to item 84, wherein the SpA-derived polypeptide or sequence A forms part of two helices, including an interconnecting loop, within the three-helix bundle protein domain. 86. The VH3-binding polypeptide according to item 84 or 85, wherein the three-helix bundle protein domain is selected from bacterial receptor domains. 87. A VH3-binding polypeptide according to any one of items 84 to 86, wherein the three-helix bundle protein domain is selected from the domain of protein A from Staphylococcus aureus or a derivative thereof. 88. The method according to claim 1, further comprising: a sequence B arranged at the C-terminus of the sequence A, the sequence B consisting of an amino acid sequence selected from iv) and v), wherein iv) and v) are selected from the following: iv) DPSX 40 SX 42 X 43 X 44 LX 46 EAX49 X 50 LNX 53 X 54 (SEQ ID NO:96) (Independently of each other, X 40 is selected from Q, T and V; X 42 is selected from A, K, L and T; X 43 is selected from N, E, A and S; X 44 is selected from L, I, and V; X 46 is selected from A, G, S and K, for example, A, S and K; X 49 is selected from K and Q, for example, K; X 50 is selected from K and R; X 53 is selected from D, E and K; and X 54 is selected from A and S); v) an amino acid sequence having at least 75% identity to the sequence defined in iv); 88. The VH3 binding polypeptide according to any one of items 2 to 87, wherein said VH3 binding polypeptide is defined as: 89. A link module sequence C, which is arranged in the following order from N-terminus to C-terminus: [Array A]-[Array B] Array A specified in any one of items 2 to 87 and array B specified in item 88, or any amino acid sequence having at least 70% identity to any sequence selected from the group consisting of residues 8 to 54 in SEQ ID NO:88, residues 8 to 54 in SEQ ID NO:89, residues 8 to 54 in SEQ ID NO:90, residues 8 to 54 in SEQ ID NO:91, residues 8 to 54 in SEQ ID NO:92, residues 11 to 57 in SEQ ID NO:93, and residues 1 to 47 in SEQ ID NO:94. 89. The VH3-binding polypeptide according to any one of items 2 to 88, consisting of: 90. A link module sequence C, which is arranged in the following order from N-terminus to C-terminus: [Array A]-[L2]-[H3] It consists of the array [Array A], [L2], and [H3]. [sequence A] is as defined in any one of items 2 to 87; and, independently of each other, [L2] DPSV (SEQ ID NO:97); DPSQ (SEQ ID NO:98); EPSQ (SEQ ID NO:99); DPST(SEQ ID NO:100); EPSV (SEQ ID NO:101); and DPSL (SEQ ID NO: 102); selected from the group consisting of; [H3] SANLLAEAKKLNDA (SEQ ID NO: 103); SKEILAEAKKLNDA (SEQ ID NO: 104); SKAILAEAKKLNDA (SEQ ID NO: 105); SANLLAEAKKLNDA (SEQ ID NO: 106); SANLLAEAKKLNES (SEQ ID NO: 107); STNVLGEAKKLNES (SEQ ID NO: 108); SANVLGEAQKLNDS (SEQ ID NO: 109); SLEILCEAKKLNDA (SEQ ID NO:110); SLEILAEAKKLNDA (SEQ ID NO: 111); and SKKILKEAKKLNKA (SEQ ID NO: 112) 90. The VH3-binding polypeptide according to any one of items 2 to 89, selected from the group consisting of: 91. [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113); [Sequence A]-DPSVSKEILAEAKKLNDA (SEQ ID NO: 114); [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115); [Sequence A]-DPSQSANLLAEAKKLNES (SEQ ID NO: 116); [Sequence A]-DPSVSLEILGEAKKLNDA (SEQ ID NO: 117); [Sequence A]-DPSVSLEILCEAKKLNDA (SEQ ID NO: 118); [Sequence A]-DPSVSLEILAEAKKLNDA (SEQ ID NO: 119); [Sequence A]-DPSVSLALLAEAKKLNDA (SEQ ID NO: 120); [Sequence A]-DPSQSANLLSEAKKLNES (SEQ ID NO: 121); [Sequence A]-DPSQSTNVLGEAKKLNES (SEQ ID NO: 122); and [Sequence A]-DPSQSANVLGEAQKLNDS (SEQ ID NO: 123); A link module sequence C selected from the group consisting of A VH3-binding polypeptide according to item 89 or 90, wherein [sequence A] is as defined in any one of items 2 to 87. 92. vi) [Sequence A]-DPSQSANLLAEAKKLNDA (SEQ ID NO: 113) (wherein [sequence A] is as defined in any one of items 2 to 87); and vii) an amino acid sequence having at least 85% identity to the sequence defined in vi); 92. The VH3 binding polypeptide according to any one of items 88 to 91, comprising a binding module sequence C selected from the group consisting of: 93. viii) [Sequence A]-DPSVSKAILAEAKKLNDA (SEQ ID NO: 115) (wherein [sequence A] is as defined in any one of items 2 to 87); and ix) an amino acid sequence having at least 85% identity to the sequence defined in viii); 92. The VH3 binding polypeptide according to any one of items 88 to 91, comprising a binding module sequence C selected from the group consisting of: 94. x) [sequence A]-DPSVSKEILAEAKKLNDA (sequence number 114) (wherein [sequence A] is as defined in any one of items 2 to 87); and xi) an amino acid sequence having at least 85% identity to the sequence defined in x); 92. The VH3 binding polypeptide according to any one of items 88 to 91, comprising a binding module sequence C selected from the group consisting of: 95. The VH3-binding polypeptide according to any one of items 2 to 95, wherein sequence vi) or viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, such as the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, such as the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56. 96. The VH3-binding polypeptide according to item 95, wherein sequence viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 41, for example the group consisting of SEQ ID NOs: 1 to 40, for example the group consisting of SEQ ID NOs: 1 to 15. 97. Sequence vi) or viii) is a group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, a group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, a group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, a group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82; For example, the VH3-binding polypeptide according to item 95, which corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54, and 56 to 81; for example, the group consisting of SEQ ID NOs: 13, 15, 54, and 56. 98. The VH3-binding polypeptide according to item 95, wherein sequence vi) or viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82; for example, the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or the group consisting of SEQ ID NOs: 15, 41, 56 and 82. 99. A VH3-binding polypeptide according to item 95, wherein sequence vi) or viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56. 100. A VH3-binding polypeptide according to item 95, wherein sequence viii) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15. 101. The VH3-binding polypeptide according to item 95, wherein sequence viii) corresponds to the group consisting of SEQ ID NOs: 15 to 41; for example, the group consisting of SEQ ID NOs: 15 to 40, or the sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 15 and 41. 102. A VH3-binding polypeptide according to item 95, wherein sequence viii) corresponds to the sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 14 and 15. 103. A VH3-binding polypeptide according to item 95, wherein sequence vi) corresponds to a sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 42, 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54, and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56. 104. The VH3-binding polypeptide according to item 95, wherein sequence vi) corresponds to the group consisting of SEQ ID NOs: 56 to 82; for example, the group consisting of SEQ ID NOs: 56 to 81, or the sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 56 and 82. 105. A VH3-binding polypeptide according to item 95, wherein sequence vi) corresponds to the sequence from positions 8 to 54 in a sequence selected from the group consisting of SEQ ID NOs: 55 and 56. 106. A VH3-binding polypeptide according to item 95, wherein sequence viii) corresponds to the sequence from positions 8 to 54 in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. 107. A VH3-binding polypeptide according to item 95, wherein sequence vi) corresponds to the sequence from positions 8 to 54 in SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. 108. A VH3-binding polypeptide according to item 83, comprising a sequence selected from the group consisting of: a sequence corresponding to residues 8 to 54 in SEQ ID NO: 83; a sequence corresponding to residues 8 to 54 in SEQ ID NO: 84; a sequence corresponding to residues 8 to 54 in SEQ ID NO: 85; a sequence corresponding to residues 11 to 57 in SEQ ID NO: 86 and residues 1 to 47 in SEQ ID NO: 87. 109. From N-terminus to C-terminus, the following order: [S1]-[binding module sequence C]-[S2] , comprising sequence 1 [S1], [binding module sequence C] and sequence 2 [S2]; [S1] or [S2] may be present or absent; [link module sequence C] is as defined in any one of items 88 to 107; and Independently of each other, [S1] is, ADNKFNK (SEQ ID NO: 124); VDAKFDK (SEQ ID NO: 125); VDNKFNK (SEQ ID NO: 126); ADAQQNKFNK (SEQ ID NO: 127); IAAKHDK (SEQ ID NO: 128); IAAQHDK (SEQ ID NO: 129); ADNNFNK (SEQ ID NO: 130); IAAKFDE (SEQ ID NO: 131); PAAKHDK (SEQ ID NO: 132); ADNAFNT (SEQ ID NO: 133); FNK; ADNRFNE (SEQ ID NO: 134); IDSKFDE (SEQ ID NO: 135); ADNRFNR (SEQ ID NO: 136); ADNKHNK (SEQ ID NO: 137); ADSKFDE (SEQ ID NO: 138); IDAKHDE (SEQ ID NO: 139); QQNKFNK (SEQ ID NO: 140); ADNKFHK (SEQ ID NO: 141); KFNK (SEQ ID NO: 142); ADNNFNR (SEQ ID NO: 143); AAAKHDK (SEQ ID NO: 144); IDNKFNK (SEQ ID NO: 145); IDAKFDE (SEQ ID NO: 146); DNNFNK (SEQ ID NO: 147); ADNKFNE (SEQ ID NO: 148); AAAQHDK (SEQ ID NO: 149); and AAAKFDE (SEQ ID NO: 150); [S2] is QAPK (SEQ ID NO: 151); QAPP (SEQ ID NO: 152); QAP; QAPR (SEQ ID NO: 153); QAPE (SEQ ID NO: 154); APK; QAPG (SEQ ID NO: 155); QAIK (SEQ ID NO: 156); and QA, 8. A VH3-binding polypeptide according to any one of items 2 to 82 and 84 to 107. 110. ADNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 157); VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158); ADNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 159); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 160); VDNKFNK-[sequence A]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 161); VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162); ADNNFNK-[sequence A]-DPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 163); ADNKHNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 164); ADNKFNK-[sequence A]-DPSQSANLLAEAAPK (SEQ ID NO: 165); IAAQHDK-[sequence A]-DPSVSLEILAEAKKLNDAQAPK (SEQ ID NO: 166); ADNKFHK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 167); FNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 168); IAAQHDK-[sequence A]-DPSVSLEILCEAKKLNDAQAPK (SEQ ID NO: 169); IDAKFDE-[sequence A]-DPSVSLALLAEAKKLNDAQAPP (SEQ ID NO: 170); DNNFNK-[sequence A]-DPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 171); VDAKFDK-[sequence A]-DPSQSANLLAEAKKLNDAQAIK (SEQ ID NO: 172); ADNRFNE-[sequence A]-DPSVSKEILAEAKKLNDAQAPE (SEQ ID NO: 173); and IDAKFDE-[sequence A]-DPSVSLSLLAEAKKLNDAQAPP (SEQ ID NO: 174); comprising a sequence selected from the group consisting of 109. A VH3-binding polypeptide according to any one of items 2 to 109, wherein [sequence A] is as defined in any one of items 2 to 87. 111. xii) VDNKFNK-[sequence A]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 158) (wherein [array A] is as defined in any one of items 2 to 87); xiii) an amino acid sequence having at least 86% identity to the sequence defined in xii); 111. The VH3-binding polypeptide according to any one of items 2 to 110, comprising a sequence selected from the group consisting of: 112. xiv) VDAKFDK-[sequence A]-DPSVSKAILAEAKKLNDAQAPK (SEQ ID NO: 162) (wherein [sequence A] is as defined in any one of items 2 to 87); and xv) an amino acid sequence having at least 86% identity to the sequence defined in xiv); 111. The VH3-binding polypeptide according to any one of items 2 to 110, comprising a sequence selected from the group consisting of: 113. The VH3-binding polypeptide according to any one of items 2 to 112, wherein sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1 to 82, such as the group consisting of SEQ ID NOs: 1 to 40 and 42 to 81, such as the group consisting of SEQ ID NOs: 1 to 15 and 42 to 56. 114. A VH3-binding polypeptide according to item 113, wherein sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1 to 41, such as the group consisting of SEQ ID NOs: 1 to 40, such as the group consisting of SEQ ID NOs: 1 to 15. 115. Sequence xii) or xiv) is the group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41, 47, 48, 51, and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51, 54, and 56 to 82 for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 13, 15, 54 and 56. 116. The VH3-binding polypeptide according to item 113, wherein sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 15 to 41 and 56 to 82; for example the group consisting of SEQ ID NOs: 15 to 40 and 56 to 81, or the group consisting of SEQ ID NOs: 15, 41, 56 and 82. 117. A VH3-binding polypeptide according to item 113, wherein sequence xii) or xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 55 and 56. 118. A VH3-binding polypeptide according to item 113, wherein sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15. 119. A VH3-binding polypeptide according to item 113, wherein sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 15 to 41; for example the group consisting of SEQ ID NOs: 15 to 40 or the group consisting of SEQ ID NOs: 15 and 41. 120. A VH3-binding polypeptide according to item 113, wherein sequence xiv) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 14 and 15. 121. A VH3-binding polypeptide according to item 113, wherein sequence xii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 42, 43 and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56. 122. The VH3-binding polypeptide according to item 113, wherein sequence xii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 56 to 82; for example the group consisting of SEQ ID NOs: 56 to 81 or the group consisting of SEQ ID NOs: 56 and 82. 123. A VH3-binding polypeptide according to item 113, wherein sequence xii) corresponds to a sequence selected from the group consisting of SEQ ID NOs: 55 and 56. 124. A VH3-binding polypeptide according to item 113, wherein sequence xiv) corresponds to SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. 125. A VH3-binding polypeptide according to item 113, wherein sequence xii) corresponds to a sequence in SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. 126. A VH3-binding polypeptide according to item 108, comprising a sequence selected from the group consisting of: a sequence corresponding to residues 1 to 58 in SEQ ID NO: 83; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 84; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 85; a sequence corresponding to residues 3 to 60 in SEQ ID NO: 86 and a sequence corresponding to residues 1 to 51 in SEQ ID NO: 87, or a sequence selected from the group consisting of: a sequence corresponding to residues 1 to 58 in SEQ ID NO: 83; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 84; a sequence corresponding to residues 1 to 58 in SEQ ID NO: 85; a sequence corresponding to residues 1 to 63 in SEQ ID NO: 86 and a sequence corresponding to residues 1 to 51 in SEQ ID NO: 87. 127. A VH3-binding polypeptide multimer, wherein each monomer of the multimer comprises a VH3-binding polypeptide independently selected from any of the VH3-binding polypeptides described in any one of items 1 to 126. 128. A VH3-binding polypeptide multimer, wherein each monomer of the multimer comprises a sequence A independently selected from any sequence A defined in any one of items 2 to 87. 129. A VH3-binding polypeptide multimer according to item 127 or 128, selected from the group consisting of a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer and decamer. 130. A VH3-binding polypeptide multimer according to any one of items 127 to 129, selected from the group consisting of dimers, tetramers, pentamers, hexamers, heptamers and octamers; for example, a dimer, a tetramer and a hexamer. 131. A VH3-binding polypeptide multimer according to any one of items 127 to 130, which is a tetramer or hexamer. 132. A VH3-binding polypeptide multimer according to any one of items 127 to 131, which is a homomer. 133. The VH3-binding polypeptide multimer according to any one of items 127 to 132, wherein the multimer comprises a sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177, for example, the sequence is SEQ ID NO: 177. 134. A VH3-binding polypeptide multimer according to any one of items 127 to 132, which is a heteromer comprising at least two or at least three different VH3-binding polypeptides, each of which is as described in any one of items 1 to 126. 135. A VH3-binding polypeptide multimer according to any one of items 127 to 131 and 134, which is a heteromer comprising at least two or at least three different sequences A, each as defined in any one of items 2 to 87. 136. A VH3-binding polypeptide multimer according to any one of items 127 to 135, further comprising at least one linker. 137. A VH3-binding polypeptide multimer according to item 136, wherein the linker comprises up to 15 amino acid residues. 138. A VH3 binding polypeptide according to any one of items 1 to 126 or a VH3 binding polypeptide multimer according to any one of items 127 to 137, comprising additional amino acids at the C-terminus and / or N-terminus. 139. The VH3-binding polypeptide or VH3-binding polypeptide multimer according to item 138, wherein the additional amino acids improve the in vitro production, purification, stabilization, or coupling of the polypeptide. 140. A VH3-binding polypeptide or VH3-binding polypeptide multimer according to item 138 or 139, wherein the additional amino acids improve coupling of the polypeptide and are selected from the group consisting of one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues. 141. A VH3-binding polypeptide multimer according to any one of items 127 to 140, wherein the VH3-binding polypeptide monomers are expressed as a fusion protein. 142. A VH3 binding polypeptide multimer according to any one of items 127 to 140, wherein the VH3 binding polypeptide monomer units are covalently linked to one another. 143. K for Interaction D The value is up to 1×10 -7 M, for example, up to 1×10 -8 M, for example, up to 1×10 -9 M, for example, up to 1×10 -10 M, for example, up to 1×10 -11 A VH3-binding polypeptide according to any one of items 1 to 126 and 138 to 140 or a VH3-binding polypeptide multimer according to any one of items 127 to 142, which is capable of binding to VH3 such that M is M. 144. K for Interaction D The value is the K D 144. A VH3-binding polypeptide according to any one of items 1 to 126, 138 to 140 and 143 or a VH3-binding polypeptide multimer according to any one of items 127 to 143, which is capable of binding to VH3 such that the VH3 binding polypeptide is at least 25%, such as at least 30%, for example at least 40%, such as at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, such as at least 90% of the value. 145. In the Fc region, 1×10 -4 Less than M, e.g. 1×10 -3 K for interactions less than M D145. A VH3-binding polypeptide according to any one of items 1 to 126, 138 to 140, 143 and 144 or a VH3-binding polypeptide multimer according to any one of items 127 to 144, which is unable to bind at a value 146. A VH3 binding polypeptide according to any one of items 1 to 126, 138 to 140, 143 and 144 or a VH3 binding polypeptide multimer according to any one of items 127 to 145, which retains at least 19%, such as at least 20%, for example at least 21%, such as at least 23%, for example at least 25%, such as at least 27%, for example at least 29%, such as at least 31%, for example at least 33%, such as at least 35%, for example at least 37%, such as at least 39%, for example at least 41%, such as at least 43%, for example at least 45%, such as at least 47%, for example at least 48%, such as at least 50% of the original VH3 binding capacity after 100 repeated binding cycles followed by washing with 0.5 M NaOH. 147. A VH3 binding polypeptide according to any one of items 1 to 126, 138 to 140 and 143 to 146 or a VH3 binding polypeptide multimer according to any one of items 127 to 146, which exhibits an alkaline wash stability of at least 35%, such as at least 40%, for example at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95% of the alkaline wash stability of SEQ ID NO: 88 or SEQ ID NO: 89 after incubation in 0.5 M NaOH, such as after repeated incubation with 0.5 M NaOH. 148. A VH3 binding polypeptide according to any one of items 1 to 126, 138 to 140 and 143 to 147 or a VH3 binding polypeptide multimer according to any one of items 127 to 147, which exhibits an alkaline wash stability within ±25% of the alkaline wash stability of SEQ ID NO: 88 or SEQ ID NO: 89, such as within ±20% of the alkaline wash stability of SEQ ID NO: 89, such as within ±15% of the alkaline wash stability of SEQ ID NO: 89, such as within ±10% of the alkaline wash stability of SEQ ID NO: 89, such as within ±5% of the alkaline wash stability of SEQ ID NO: 88 or SEQ ID NO: 89, after incubation in 0.5 M NaOH, such as after repeated incubation with 0.5 M NaOH. 149. A polynucleotide encoding a VH3-binding polypeptide according to any one of items 1 to 126, 138 to 140 and 143 to 148 or a VH3-binding polypeptide multimer according to any one of items 127 to 141 and 143 to 148. 150. An expression vector comprising the polynucleotide according to item 149. 151. A host cell comprising the expression vector according to item 150. 152. A method for producing a VH3-binding polypeptide according to any one of items 1 to 126, 138 to 140 and 143 to 148 or a VH3-binding polypeptide multimer according to any one of items 127 to 141 and 143 to 148, comprising: - culturing a host cell according to item 151 under conditions allowing expression of said polypeptide or multimer from said expression vector, and - isolating said polypeptide or multimer. The method includes: 153. A separation matrix comprising a VH3 binding polypeptide according to any one of items 1 to 126, 138 to 140 and 143 to 148 or a VH3 binding polypeptide multimer according to any one of items 127 to 148 coupled to a solid support. 154. The separation matrix according to item 153, wherein the VH3-binding polypeptide multimer is described in any one of items 127 to 148. 155. A separation matrix according to item 153 or 154, wherein the VH3-binding polypeptide multimer is a tetramer or hexamer. 156. The VH3-binding polypeptide is selected from the group consisting of SEQ ID NOs: 1 to 87, for example, the group consisting of SEQ ID NOs: 1 to 82; for example, the group consisting of SEQ ID NOs: 1, 2, 4 to 43, and 45 to 82; For example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41, 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41, 47, 48, 51 ..., 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41, 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 41, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 13, 15 to 40, 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 13, 15, 54 and 56. The separation matrix according to any one of items 153 to 155, comprising a sequence selected from the group consisting of SEQ ID NOs: 13, 15, 54 and 56. 157. The separation matrix according to any one of items 153 to 156, wherein the VH3-binding polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 41; for example, the group consisting of SEQ ID NOs: 1, 2, 4 to 41; for example, the group consisting of SEQ ID NOs: 5 to 7, 9 to 11, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13 to 41; for example, the group consisting of SEQ ID NOs: 6, 7, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 6, 10, 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 41; for example, the group consisting of SEQ ID NOs: 13, 15 to 40; for example, the group consisting of SEQ ID NOs: 13 and 15. 158. The separation matrix according to any one of items 153 to 156, wherein the VH3-binding polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 42 to 82; for example, the group consisting of SEQ ID NOs: 42, 43 and 45 to 82; for example, the group consisting of SEQ ID NOs: 46 to 48, 50 to 52 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51 and 54 to 82; for example, the group consisting of SEQ ID NOs: 47, 48, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 47, 51, 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 82; for example, the group consisting of SEQ ID NOs: 54 and 56 to 81; for example, the group consisting of SEQ ID NOs: 54 and 56. 159. A separation matrix according to any one of items 153 to 157, wherein the VH3-binding polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. 160. A separation matrix according to any one of items 153 to 156 and 158, wherein the VH3-binding polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56. 161. A separation matrix according to any one of items 153 to 156, wherein the VH3-binding polypeptide comprises the sequence of SEQ ID NO: 41 or SEQ ID NO: 82. 162. A separation matrix according to any one of items 153 to 157 and 159, comprising a VH3-binding multimer selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177. 163. A separation matrix according to any one of items 153 to 162, wherein the solid support comprises a polymer selected from the group consisting of polyhydroxypolymers, such as polysaccharides; polyvinyl alcohols, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, and polymethacrylamides. 164. The separation matrix according to item 163, wherein the polymer is a polysaccharide, preferably a polysaccharide selected from dextran, starch, cellulose, pullulan, agar and agarose. 165. The separation matrix according to any one of items 153 to 164, wherein the solid support is in fibrous, beaded or particulate form. 166. The separation matrix according to any one of items 153 to 165, wherein the VH3-binding polypeptide is coupled to the solid support by covalent or non-covalent bonds. 167. a) contacting a liquid sample containing an immunoglobulin or a fragment thereof with a separation matrix according to any one of items 153 to 166. 2. A method for isolating an immunoglobulin or a fragment thereof, comprising: 168. b) washing the separation matrix with a washing solution; and c) eluting the immunoglobulin or fragment thereof from the separation matrix using an elution solution, and optionally d) washing the separation matrix with a washing solution Item 168. The method of item 167, further comprising: 169. The method according to item 167 or 168, wherein the fragment is selected from a Fab fragment, a Fab' fragment, an F(ab')2, an scFab fragment, an Fv fragment, an scFv fragment, a diabody, a triabody, and a minibody. 170. The method of any one of items 167 to 169, wherein the immunoglobulin or fragment thereof is an asymmetric immunoglobulin or fragment thereof. 171. The method according to item 170, wherein the asymmetric immunoglobulin or fragment thereof is a multispecific immunoglobulin or fragment thereof, such as at least a bispecific or trispecific immunoglobulin or fragment thereof. 172. The method according to any one of items 167 to 171, wherein the separation matrix comprises a VH3-binding polypeptide according to SEQ ID NO: 14 or a multimer comprising SEQ ID NO: 14, and the pH level of elution is above 4, for example in the range of approximately 4.10 to 4.20, for example approximately 4.15. 173. The method according to any one of items 167 to 171, wherein the separation matrix comprises a VH3-binding polypeptide according to SEQ ID NO: 15 or a multimer comprising SEQ ID NO: 15, and the pH level of elution is below 4, for example in the range of approximately 3.7 to 3.80, for example approximately 3.75. 174. a) providing a liquid sample comprising a mixture of at least one symmetric immunoglobulin or fragment thereof and at least one asymmetric immunoglobulin or fragment thereof; b) contacting the liquid sample with a separation matrix, wherein the symmetric immunoglobulin or fragment thereof and the asymmetric immunoglobulin or fragment thereof bind to the separation matrix with different binding strengths, the separation matrix being defined as in claim 14; c) eluting said immunoglobulins or fragments thereof from the separation matrix, wherein said symmetric immunoglobulins or fragments thereof and said asymmetric immunoglobulins or fragments thereof are at least partially eluted separately from each other, and optionally d) washing the separation matrix with a washing solution 2. A method for isolating an asymmetric immunoglobulin or a fragment thereof, comprising: 175. Step c): c) eluting said immunoglobulins or fragments thereof from the separation matrix by a pH gradient or by stepwise elution at least two different pH levels, whereby said symmetric immunoglobulins or fragments thereof and said asymmetric immunoglobulins or fragments thereof are at least partially eluted at different pH levels. Item 175. The method according to item 174, involving 176. A method for isolating an asymmetric immunoglobulin or a fragment thereof according to item 174 or 175, wherein the asymmetric immunoglobulin or a fragment thereof comprises only one VH3 region, and the symmetric immunoglobulin or a fragment thereof comprises at least two VH3 regions or no VH3 region. 177. A method for isolating an asymmetric immunoglobulin or a fragment thereof according to any one of items 174 to 176, wherein the separation matrix is ​​as defined in any one of items 153 to 166. 178. 25% or less, such as 20% or less, such as 15% or less, such as 10% or less, of asymmetric immunoglobulins or fragments thereof are present in the same eluate as symmetric immunoglobulins or fragments thereof. 178. A method for isolating an asymmetric immunoglobulin or a fragment thereof according to any one of items 174 to 177. 179. A method for isolating an asymmetric immunoglobulin or a fragment thereof according to any one of items 174 to 178, wherein the asymmetric immunoglobulin or a fragment thereof is a multispecific immunoglobulin or a fragment thereof, such as at least a bispecific or trispecific immunoglobulin or a fragment thereof. 180. A VH3-binding polypeptide or any domain thereof derived from Staphylococcus aureus protein A (SpA), comprising at position X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26 The positions in the alignment with SEQ ID NO: 89 corresponding to the positions are, independently of one another, one of the following: X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11 is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G; A VH3 binding polypeptide or any domain thereof, 181. A VH3-binding polypeptide or any domain thereof derived from Staphylococcus aureus protein A (SpA), comprising at position X9, X 10 Place, X 11 Place, X 13 Place, X 14 Place, X 17 Place, X 18 Rank and X 26 The positions in the alignment with SEQ ID NO: 88 corresponding to the positions are, independently of one another, one of the following: X9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G; A VH3 binding polypeptide or any domain thereof,

Claims

1. A VH3-binding polypeptide derived from Staphylococcus aureus protein A (SpA), or any domain thereof, which has binding affinity to the VH3 region of trastuzumab and has a lower binding affinity to the Fc region of trastuzumab compared to the binding affinity of SEQ ID NO: 88 to the same Fc region, and is alkaline wash stable.

2. Array A, wherein array A is: i)X 8 X 9 X 10 X 11 AX 13 X 14 X 15 X 16 X 17 X 18 X 19 PNLX 23 X 24 X 25 X 26 R X 28 X 29 FIQSLX 35 X 36 (SEQ ID NO:95) (Independently of each other, X 8 is selected from E, D and A; X 15 is selected from E and Q; X 16 is selected from I and V; X 19 is selected from L and M; X 23 is selected from N and T; X 24 is selected from A and E, for example, E; X 25 is selected from D and E, for example E; X 35 is selected from K, R and H; X 36 is selected from D and H); ii) an amino acid sequence having at least 83% identity to the sequence defined under i); iii) an amino acid sequence having at least 70% identity to any sequence selected from the group consisting of residues 8 to 36 in SEQ ID NO:88, residues 8 to 36 in SEQ ID NO:89, residues 8 to 36 in SEQ ID NO:90, residues 8 to 36 in SEQ ID NO:91, residues 8 to 36 in SEQ ID NO:92, residues 11 to 39 in SEQ ID NO:93, and residues 1 to 29 in SEQ ID NO:

94. and wherein the amino acid sequence is selected from i), ii), and iii) defined as follows: Further, in each of i), ii) and iii), independently of one another, X 9 is selected from Q, Y and A; X 10 is selected from Q and Y; X 11 is selected from T, E and R; X 13 is selected from L, E, R, A and Q; X 14 is selected from L, E, R, A, Q, and W; X 17 is selected from A, H and L; X 18 is selected from R, L and H; X 26 is selected from Q and S; X 28 is selected from N and A; and X 29 is selected from A and G; 2. A VH3 binding polypeptide as described in claim 1.

3. X 9 is A and X 11 A VH3 binding polypeptide as described in claim 2, wherein is R.

4. X 9 X 10 X 11 X 13 X 14 is selected from the group consisting of AQRLA and AYRLA; for example, X 9 X 10 X 11 X 13 X 14 4. A VH3 binding polypeptide as claimed in claim 2 or 3, wherein said VH3 binding polypeptide is AYRLA.

5. X 17 X 18 5. A VH3 binding polypeptide according to any one of claims 2 to 4, wherein is AR.

6. 6. A VH3-binding polypeptide according to any one of claims 2 to 5, wherein sequence i) corresponds to the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 82.

7. The method further comprises: disposing sequence B at the C-terminus of sequence A; and sequence B is an amino acid sequence selected from iv) and v), wherein iv) and v) are selected from the following: iv) DPSX 40 SX 42 X 43 X 44 LX 46 EAX 49 X 50 LNX 53 X 54 (SEQ ID NO:96) (Independently of each other, X 40 is selected from Q, T and V; X 42 is selected from A, K, L and T; X 43 is selected from N, E, A and S; X 44 is selected from L, I, and V; X 46 is selected from A, G, S and K, for example, A, S and K; X 49 is selected from K and Q, for example, K; X 50 is selected from K and R; X 53 is selected from D, E and K; and X 54 is selected from A and S); v) an amino acid sequence having at least 75% identity to the sequence defined in iv); 7. A VH3 binding polypeptide according to any one of claims 2 to 6, defined as follows:

8. The link module sequence C includes, from the N-terminus to the C-terminus, the following order: [Array A]-[Array B] A sequence as defined in any one of claims 2 to 6 and a sequence as defined in claim 7, or any amino acid sequence having at least 70% identity to any sequence selected from the group consisting of residues 8-54 in SEQ ID NO:88, residues 8-54 in SEQ ID NO:89, residues 8-54 in SEQ ID NO:90, residues 8-54 in SEQ ID NO:91, residues 8-54 in SEQ ID NO:92, residues 11-57 in SEQ ID NO:93, and residues 1-47 in SEQ ID NO:

94.

8. A VH3 binding polypeptide according to any one of claims 2 to 7, consisting of:

9. vi) [Sequence A] -DPSQSANLLAEAKKLNDA (SEQ ID NO: 113) (wherein [sequence A] is as defined in any one of claims 2 to 6); vii) an amino acid sequence having at least 85% identity to the sequence defined in vi); viii) [Sequence A] -DPSVSKAILAEAKKLNDA (SEQ ID NO: 115) (wherein [sequence A] is as defined in any one of claims 2 to 6); ix) an amino acid sequence having at least 85% identity to the sequence defined in viii); x) [Sequence A] -DPSVSKEILAEAKKLNDA (SEQ ID NO: 114) (wherein [sequence A] is as defined in any one of claims 2 to 6); and xi) an amino acid sequence having at least 85% identity to the sequence defined in x); 9. A VH3 binding polypeptide according to any one of claims 2 to 8, comprising a binding module sequence C selected from the group consisting of:

10. 10. A VH3-binding polypeptide according to any one of claims 2 to 9, wherein sequence vi) or viii) corresponds to a sequence from position 8 to 54 in a sequence selected from the group consisting of SEQ ID NO: 1 to 82, such as the group consisting of SEQ ID NO: 1 to 41, such as the group consisting of SEQ ID NO: 1 to 40, such as the group consisting of SEQ ID NO: 1 to 15, such as the group consisting of SEQ ID NO: 13 to 15, such as SEQ ID NO:

15.

11. A VH3 binding polypeptide multimer, wherein each monomer of the multimer comprises a VH3 binding polypeptide independently selected from any one of the VH3 binding polypeptides described in any one of claims 1 to 10, and preferably the multimer is a tetramer or hexamer.

12. 12. A VH3 binding polypeptide multimer as described in claim 11, wherein the multimer comprises an sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177, for example, the sequence is SEQ ID NO:

177.

13. 13. A VH3 binding polypeptide according to any one of claims 1 to 10 or a VH3 binding polypeptide multimer according to claim 11 or 12, which exhibits an alkaline wash stability of at least 35%, such as at least 40%, for example at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95% of the alkaline wash stability of SEQ ID NO: 88 after incubation in 0.5 M NaOH.

14. A separation matrix comprising a VH3 binding polypeptide described in any one of claims 1 to 10 and 13 or a VH3 binding polypeptide multimer described in any one of claims 11 to 13 coupled to a solid support, preferably in fibrous, bead or particulate form.

15. a) contacting a liquid sample containing immunoglobulins or fragments thereof with a separation matrix according to claim 14 2. A method for isolating an immunoglobulin or a fragment thereof, comprising:

16. a) providing a liquid sample comprising a mixture of at least one symmetric immunoglobulin or fragment thereof and at least one asymmetric immunoglobulin or fragment thereof; b) contacting the liquid sample with a separation matrix, wherein the symmetric immunoglobulin or fragment thereof and the asymmetric immunoglobulin or fragment thereof bind to the separation matrix with different binding strengths, the separation matrix being defined as in claim 14; c) eluting said immunoglobulins or fragments thereof from the separation matrix, wherein said symmetric immunoglobulins or fragments thereof and said asymmetric immunoglobulins or fragments thereof are at least partially eluted separately from each other, and optionally d) washing the separation matrix with a washing solution 2. A method for isolating an asymmetric immunoglobulin or a fragment thereof, comprising:

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