Scaffold Proteins

Abstract

The present invention provides polypeptides, such as Affimer polypeptides, comprising an amino acid sequence having at least 80% identity to amino acid residues 1-11, 13-15, 17-19, 21-25, 27-28, 35-37, 39, 41, 43-44, 46-47, 49-50, 52-53, 55-58, 63-64, 66, 68-82, 84-85, and 87-98 of SEQ ID NO: 1, wherein the polypeptides include T51L, ... V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34D , T34P, A40V, Q42D, T45I, T45V, V48E, V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V) , (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G , N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), ( The present invention relates to polypeptides characterized by comprising one or more mutations selected from the group consisting of (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E). The present invention also relates to various methods and nucleic acids.

Description

[Background technology]

[0001] WO 2009 / 136182 discloses mutations of the prior art scaffold STM using D48L and G50S, which are disclosed to result in increased expression in bacterial systems (page 22, lines 12-13).

[0002] WO 2009 / 136182 and WO 2006 / 131749 each previously disclosed mutations at the V48 site of wild-type Stefin A, in particular the V48D mutation (WO 2006 / 131749, page 35, line 25), which are useful for abolishing domain-swap dimerization.

[0003] The co-mutation of E78A and L80R has been disclosed for the scaffold SQT in WO 2009 / 136182, as well as the co-mutation of L82R and T83S in the SQM scaffold (paragraphs spanning pages 22-23 of WO 2009 / 136182). These co-mutation pairs have been shown to exhibit high expression in Escherichia coli (E. coli).

[0004] WO 2009 / 136182 discloses modified stefin A scaffold proteins. In particular, the disclosure in this document focuses on "position 4 mutations" corresponding to the G4 site of stefin A or the W4 site of STM. In particular, this document teaches the particular advantages of G4R mutants. This document does not provide any teaching regarding changes in the thermal stability of the scaffold proteins. Thermal stability is mentioned in only one paragraph in this document as one of several properties that can be measured to assess the resistance of the scaffold protein to modification by a target peptide (WO 2009 / 136182, page 13, first paragraph).

[0005] WO 2006 / 131749 discloses the use of stefin A as a scaffold protein and discloses several mutations useful for ensuring the biological neutrality of the scaffold. The document discloses a thermal stability assay (page 32, first paragraph) and presents data on the thermal stability of STM (page 35, lines 9-12). Thermal stability in this document is disclosed merely as a property that can be measured to assess the resistance of a scaffold protein to modification by a target peptide (see paragraphs covering pages 6-7 of WO 2006 / 131749). The document does not provide any teaching regarding altering or modulating thermal stability.

[0006] WO 2014 / 125290 discloses scaffold proteins derived from plant cystatins. In particular, the document discloses three preferred synthetic proteins as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 of WO 2014 / 125290. The document discloses the measurement of the thermal stability of these preferred scaffolds by differential scanning calorimetry (page 48, lines 31-32, and Figure 7a). Nowhere in the document is there any disclosure of mutations made to affect thermal stability. In fact, the inventors do not comment on any method of influencing the thermal stability of their scaffold proteins. They assert that the thermal stability of their preferred scaffolds is "high," but attribute this to the inherent compactness of their scaffolds: "The compact nature of the scaffold, which is more pronounced than seen in other structures of stefins or cystatins seems likely to contribute to its high thermal stability." (International Publication No. 2014 / 125290, page 53, lines 17-19).

[0007] There is also no teaching in this document on how to modulate or change thermostability. Furthermore, mutations are discussed in this document only in relation to prior art mutations or the need to use trimeric insertions or deletions to avoid frameshift mutations. The scientific focus of this document is on designing consensus sequences derived from plant cystatin proteins, and there is no disclosure of sequence manipulation to affect thermostability. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2009 / 136182 Brochure [Patent Document 2] International Publication No. 2006 / 131749 Brochure [Patent Document 3] International Publication No. 2014 / 125290 Brochure Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have studied the Stefin A scaffold protein with the goal of improving its properties, such as its thermal stability. Proceeding based on insights into protein structure and function, the inventors designed various changes and substitutions to improve those properties. This has led to a variety of unexpected results, including, for example, unpredicted anomalies (e.g., the Q42D / E anomaly, discussed in detail below) in which the protein might have been predicted to behave in a particular way, but for which objective knowledge contradicted that prediction. The result of this considerable research and intellectual effort is a comprehensive teaching of how to alter the thermal stability of the Stefin A protein and how these changes in thermal stability benefit its use as a scaffold protein. [Means for solving the problem]

[0010] Accordingly, the present invention provides a polypeptide, such as an Affimer (Affimer) polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1-11, 13-15, 17-19, 21-25, 27-28, 35-37, 39, 41, 43-44, 46-47, 49-50, 52-53, 55-58, 63-64, 66, 68-82, 84-85, and 87-98 of SEQ ID NO: 1, For SEQ ID NO: 1, T51L, T51V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q2 6E, E29M, T31K, N32D, N32H, T34V, T34R, T34D, T34P, A40V, Q42D, T45I, T45V, V48E , V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD 61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) The present invention relates to a polypeptide characterized by comprising one or more mutations selected from the group consisting of:

[0011] The present invention provides a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1, For SEQ ID NO: 1, T51L, T51V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q2 6E, E29M, T31K, N32D, N32H, T34V, T34R, T34D, T34P, A40V, Q42D, T45I, T45V, V48E , V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD 61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) The present invention relates to a polypeptide characterized by comprising one or more mutations selected from the group consisting of:

[0012] The present invention provides a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1 to 98 of SEQ ID NO: 1, For SEQ ID NO: 1, T51L, T51V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q2 6E, E29M, T31K, N32D, N32H, T34V, T34R, T34D, T34P, A40V, Q42D, T45I, T45V, V48E , V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD 61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) The present invention relates to a polypeptide characterized by comprising one or more mutations selected from the group consisting of:

[0013] Suitably, said one or more mutations to SEQ ID NO: 1 are: T51L, T51V, M65V, N32G, A59I, E29M, T34V, T34R, T45I, T45V, T51F, A59L, L67I, (E29K , K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G6 0N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) and preferably said polypeptide has a Tm higher than the Tm of SEQ ID NO:1.

[0014] Suitably, said one or more mutations to SEQ ID NO: 1 are: L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q26E, T31K, N32D, N32H, T34D , T34P, A40V, Q42D, V48E, V48G, V48A, T51A, (V20I, L38A), (V20L, L38A), (V20 I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) and preferably said polypeptide has a Tm lower than the Tm of SEQ ID NO:1.

[0015] Another aspect of the present invention is a compound of the general formula: MIP-Xaa1-GLSEAKPA 12 TPEI 16 QEIV 20 DKVKPQ 26 LEE 29 K 30 T 31 N 32 E 33 T 34 YGKL 38 EA 40 VQ 42 YKT 45 QVV 48 A-(Xaa)n-Xaa2-T 51 NYY 54 IKVRA 59 G 60 D 61 N 62 KYM 65 HL 67 KVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLT 83 GYQ 86 VDKNKDDELTGF (In the sequence, Xaa, individually for each occurrence, is an amino acid residue; n and m are each independently an integer of 3 to 20; Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser, or Thr; Xaa3 is Arg, Lys, Asn, Gln, Ser, Thr; Xaa4 is Gly, Ala, Val, Ser, or Thr; Xaa5 is Ala, Val, Ile, Leu, Gly, or Pro; Xaa6 is Gly, Ala, Val, Asp, or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg, or Lys), At least one of the following amino acid positions is selected from the alternative amino acid residues listed: A 12 is Ala or an alternative amino acid residue selected from Val, Ile or Leu, preferably Ile or Val; I 16 is Ile or an alternative amino acid residue selected from Val or Leu, preferably Leu; V 20 is Val or an alternative amino acid residue selected from Ala, Ile or Leu, preferably Ile or Leu; Q 26 is Gln or an alternative amino acid residue selected from Asp or Glu, preferably Glu; E 29 is Glu or an alternative amino acid residue selected from Asp or Met, preferably Met; K 30 is Lys or an alternative amino acid residue selected from Arg, His, Glu or Asp, preferably Glu; T31 is Thr or an alternative amino acid residue selected from Ser, Arg or Lys, preferably Lys; N 32 is Asn or an alternative amino acid residue selected from Gly, Asp, Glu or His, preferably Gly, Asp or His; E 33 is Glu or an alternative amino acid residue selected from Arg, His, Lys or Asp, preferably Lys; T 34 is Thr or an alternative amino acid residue selected from Ser, Ala, Val, Ile, Leu, Arg, Lys, Asp, Glu or Pro, preferably Val, Arg, Asp or Pro; L 38 is Leu or an alternative amino acid residue selected from Gly, Ala or Val, preferably Ala or Val; A 40 is Ala or an alternative amino acid residue selected from Gly, Val, Leu or Ile, preferably Ile or Val; Q 42 is Gln or an alternative amino acid residue selected from Asp, Glu or Asn, preferably Asp; T 45 is Thr or an alternative amino acid residue selected from Ser, Ala, Val, Ile or Leu, preferably Ile or Val; V 48 is Val or an alternative amino acid residue selected from Gly, Ala, Ile, Leu, Glu or Asp, preferably Gly, Ala or Glu; T 51 is Thr or an alternative amino acid residue selected from Gly, Ala, Val, Ile, Leu, Ser or Phe, preferably Phe, Ala, Val or Leu; Y 54is Tyr or an alternative amino acid residue selected from Asp or Glu, preferably Asp; A 59 is Ala or an alternative amino acid residue selected from Gly, Val, Ile or Leu, preferably Ile, Leu or Val; G 60 is Gly or an alternative amino acid residue selected from Asn, Gln, Gly or Pro, preferably Asn or Pro; D 61 is Asp or an alternative amino acid residue selected from ΔD61 (absent), Gly, Pro, Glu, Asn or Gln, preferably ΔD61 (absent), Gly, Pro or Asn; N 62 is Asn or an alternative amino acid residue selected from Gln, Lys, Arg, His, Gly or Pro, preferably Lys, Pro or Gly; M 65 is Met or an alternative amino acid residue selected from Ala, Val, Ile, Leu, preferably Val; L 67 is Leu or an alternative amino acid residue selected from Ala, Val or Ile, preferably Ile; T 83 is Thr or an alternative amino acid residue selected from Ser, Asp or Glu, preferably Asp; Q 86 is Gln or an alternative amino acid residue selected from Asn, Glu or Asp, preferably Glu.

[0016] In one aspect, the present invention provides a polypeptide as described above, further comprising at least one heterologous peptide insertion, The heterologous peptide insertion is inserted at one of the following positions relative to SEQ ID NO: 1: a) 47-<foreign peptide>-55 b) 46-<foreign peptide>-54 c) 46-<foreign peptide>-50 d) 48-<foreign peptide>-50 e) 49-<foreign peptide>-51 f) 50-<heterologous peptide>-52 g) 66-<foreign peptide>-85 h) 67-<foreign peptide>-84 i) 70-<foreign peptide>-74 j) 72-<foreign peptide>-74 k) 71-<foreign peptide>-73 l) 72-<foreign peptide>-81 m) 73-<foreign peptide>-80 n) 79-<foreign peptide>-81 o) 80-<foreign peptide>-81 p)82-<foreign peptide>-83 q) 72-<foreign peptide>-77 r) 73-<foreign peptide>-78 s)74-<foreign peptide>-79 t) 4-<foreign peptide>-5 The present invention relates to a polypeptide comprising:

[0017] Suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (a) to (f) and a second heterologous peptide insertion at any of positions (g) to (s).

[0018] Suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (a) to (f) and a second heterologous peptide insertion at position (t).

[0019] Suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (g) to (s) and a second heterologous peptide insertion at position (t).

[0020] Suitably, the polypeptide comprises three heterologous peptide insertions: a first heterologous peptide insertion at any of positions (a) to (f), a second heterologous peptide insertion at any of positions (g) to (s), and a third heterologous peptide insertion at position (t).

[0021] In one aspect, the invention provides a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1-11, 13-15, 17-19, 21-25, 27-28, 35-37, 39, 41, 43-44, 46-47, 49-50, 52-53, 55-58, 63-64, 66, 68-82, 84-85, and 87-98 of SEQ ID NO: 1, and comprising at least one heterologous peptide insertion; The peptide has the following structure relative to SEQ ID NO: 1: M65I, T51I, T51L, T51V, M65V, A59V, N32G, A59I, L38A, V20A, V20I, A40I, L38V, G50S, L38F, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34K, T34D, T34P, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, T51F, T51A, A59L, K63R, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D , Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N 62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) , (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) and wherein the mutation comprises one or more mutations selected from the group consisting of: The heterologous peptide insertion is inserted at one of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 The present invention relates to a polypeptide comprising:

[0022] In one aspect, the invention provides a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising at least one heterologous peptide insertion, The polypeptide has a sequence similar to that of SEQ ID NO: 1: M65I, T51I, T51L, T51V, M65V, A59V, N32G, A59I, L38A, V20A, V20I, A40I, L38V, G50S, L38F, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34K, T34D, T34P, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, T51F, T51A, A59L, K63R, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D , Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N 62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) , (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) and wherein the mutation comprises one or more mutations selected from the group consisting of: The heterologous peptide insertion is inserted at one of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 The present invention relates to a polypeptide comprising:

[0023] In one aspect, the invention provides a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1-98 of SEQ ID NO: 1, and comprising at least one heterologous peptide insertion, The polypeptide has a sequence similar to that of SEQ ID NO: 1: M65I, T51I, T51L, T51V, M65V, A59V, N32G, A59I, L38A, V20A, V20I, A40I, L38V, G50S, L38F, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34K, T34D, T34P, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, T51F, T51A, A59L, K63R, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D , Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N 62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) , (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) and wherein the mutation comprises one or more mutations selected from the group consisting of: The heterologous peptide insertion is inserted at one of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 The present invention relates to a polypeptide comprising:

[0024] Suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (d) to (f) and a second heterologous peptide insertion at any of positions (q) to (s).

[0025] Suitably, said one or more mutations to SEQ ID NO: 1 are: M65I, T51I, T51L, T51V, M65V, A59V, N32G, A59I, E29M, T34V, T34R, T34K, Q42E, T45I, T45V, T51F , A59L, K63R, L67I, N90T, (E29K, K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G6 0N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) and preferably said polypeptide has a Tm higher than the Tm of SEQ ID NO:1.

[0026] Suitably, said one or more mutations to SEQ ID NO: 1 are: L38A, V20A, V20I, A40I, L38V, G50S, L38F, A12I, A12V, I16L, V20L, Q26E, T31K, N32D, N32H, T34D, T34P, A40V, Q42D, V48E, V48D, V48G, V48A, V48L, T51A, (V20I, L38A), (V20 L, L38A), (V20I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60 P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) is selected from the group consisting of Preferably, the polypeptide has a Tm lower than the Tm of SEQ ID NO:1.

[0027] Suitably, said one or more mutations to SEQ ID NO: 1 are: T51L, T51V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q2 6E, E29M, T31K, N32D, N32H, T34V, T34R, T34D, T34P, A40V, Q42D, T45I, T45V, V48E , V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD 61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) is selected from the group consisting of:

[0028] Suitably, said one or more mutations to SEQ ID NO: 1 are: T51L, T51V, M65V, N32G, A59I, E29M, T34V, T34R, T45I, T45V, T51F, A59L, L67I, (E29K , K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G6 0N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) and preferably said polypeptide has a Tm higher than the Tm of SEQ ID NO:1.

[0029] Suitably, said one or more mutations to SEQ ID NO: 1 are: L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q26E, T31K, N32D, N32H, T34D , T34P, A40V, Q42D, V48E, V48G, V48A, T51A, (V20I, L38A), (V20L, L38A), (V20 I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) and preferably said polypeptide has a Tm lower than the Tm of SEQ ID NO:1.

[0030] In one aspect, the present invention relates to a polypeptide as described above, which has a Tm higher than the Tm of SEQ ID NO:1.

[0031] In one aspect, the present invention relates to a polypeptide as described above, which has a Tm lower than the Tm of SEQ ID NO:1.

[0032] Suitably, the polypeptide further comprises one or more mutations relative to SEQ ID NO: 1 selected from the group consisting of G4R, G4W, V48D, V48E, G50S, Y35W, Y43W, Y53W, Y54W, Y64W, F70W, Y85W, F98W, (K71N S72G L73P) and (E78A L80R).

[0033] Suitably, the polypeptide comprises five or fewer mutations relative to SEQ ID NO:1.

[0034] Suitably, the five or fewer mutations are selected from the group consisting of Y35W, N32G, V48D, M65I, Q42E and T51L.

[0035] In one embodiment, the invention relates to the aforementioned polypeptide, wherein said five or fewer mutations are selected from the group consisting of N32G, V48D, M65I, Q42E and T51L.

[0036] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: i) N32G V48D ii) N32G V48D M65I iii) N32G V48D M65I T51L iv) N32G V48D M65I Q42E v) N32G V48D M65I Q42E T51L The present invention relates to the above polypeptides having each mutation of one of the groups:

[0037] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: i) N32G V48D ii) N32G V48D M65I iii) N32G V48D M65I T51L iv) N32G V48D M65I Q42E v) N32G V48D M65I Q42E T51L and no further mutations relative to SEQ ID NO: 1.

[0038] In one embodiment, the invention relates to a polypeptide as described above, which has each of the mutations iv) N32G, V48D, M65I and Q42E.

[0039] In one embodiment, the invention relates to a polypeptide as described above, having each of the mutations iv) N32G, V48D, M65I and Q42E and no further mutations relative to SEQ ID NO: 1.

[0040] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a)Y35W N32G V48D M65I Q42E T51L(A59V ΔD61)(E29K K30E E33K) b)Y35W N32G V48D M65I Q42E T51L(A59V G60N ΔD61 N62G)(E29K K30E E33K) The present invention relates to the above polypeptides having each mutation of one of the groups:

[0041] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a)Y35W N32G V48D M65I Q42E T51L(A59V ΔD61)(E29K K30E E33K) b)Y35W N32G V48D M65I Q42E T51L(A59V G60N ΔD61 N62G)(E29K K30E E33K) and no further mutations relative to SEQ ID NO: 1.

[0042] In one embodiment, the invention relates to the aforementioned polypeptide, having each of the mutations b) Y35W N32G V48D M65I Q42E T51L (A59V G60N ΔD61 N62G) (E29K K30E E33K).

[0043] In one embodiment, the invention relates to the above-mentioned polypeptide having each of the mutations b) Y35W N32G V48D M65I Q42E T51L (A59V G60N ΔD61 N62G) (E29K K30E E33K) and no further mutations relative to SEQ ID NO: 1.

[0044] Suitably, the heterologous peptide is between 6 and 36 amino acids in length.

[0045] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: - a polypeptide as defined above, - a secretory signal sequence, a peptide linker sequence, an affinity tag, a transmembrane domain, a cell surface retention sequence, a substrate recognition sequence for post-translational modification, a multimerization domain for generating a multimeric structure of a protein that aggregates through protein-protein interactions, a half-life extending polypeptide moiety, a polypeptide sequence that alters the tissue localization and antigen-binding site of the antibody, one or more of the above additional polypeptides that bind to the same or different targets, and one or more additional Affimer polypeptide sequences that bind to the same or different targets. and one or more additional amino acid sequences selected from the group consisting of: The present invention relates to a fusion protein comprising:

[0046] Suitably, the fusion protein comprises one or more half-life extending polypeptide moieties selected from the group consisting of an Fc domain or a portion thereof, an albumin protein or a portion thereof, an albumin binding polypeptide moiety, transferrin or a portion thereof, a transferrin binding polypeptide moiety, fibronectin or a portion thereof, and a fibronectin binding polypeptide moiety.

[0047] Suitably the Fc domain or portion thereof retains FcN ​​binding.

[0048] Suitably the Fc domain or part thereof is from IgA, IgD, IgE, IgG and IgM, or a subclass (isotype) thereof, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0049] Suitably, the Fc domain or portion thereof retains an effector function selected from C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of the B-cell receptor, or a combination thereof.

[0050] Suitably, the half-life extending polypeptide moiety increases the serum half-life of the protein by at least 5-fold compared to the absence of the protein.

[0051] Suitably the polypeptide is an affimer or an affimer polypeptide. Affimers / affimer polypeptides are described in more detail below.

[0052] In one aspect, the present invention relates to a nucleic acid comprising a nucleotide sequence encoding the above-described polypeptide.

[0053] In one aspect, the present invention relates to a vector comprising the above-described nucleic acid.

[0054] In one aspect, the invention relates to a library comprising a population of polypeptides as described above, wherein at least two individual polypeptides within said population comprise a different heterologous peptide insert.

[0055] In one aspect, the present invention relates to a library comprising a population of nucleic acids, said nucleic acids comprising nucleotide sequences encoding a population of polypeptides as defined above. Suitably, each nucleic acid encodes a single polypeptide as defined above.

[0056] In one aspect, the present invention relates to a host cell comprising the above-mentioned polypeptide, the above-mentioned nucleic acid or the above-mentioned library.

[0057] In one aspect, the present invention relates to a polypeptide as described above for use in medicine.

[0058] In one aspect, the present invention provides a method for identifying a polypeptide capable of binding to a desired structure, comprising the steps of: (i) providing a polypeptide as described above containing a heterologous peptide insertion; (ii) contacting the polypeptide with the desired structure; (iii) monitoring the association of said polypeptide with said desired structure; Including, The present invention relates to a method in which the association of a polypeptide with a desired structure identifies the peptide as a candidate peptide capable of binding to the structure.

[0059] In one aspect, the present invention relates to the use of the above-mentioned polypeptides as scaffold proteins.

[0060] More suitably, the present invention provides a method for the preparation of a heterologous peptide, wherein the heterologous peptide insertion is inserted at one of the following positions relative to SEQ ID NO: 1: d) 48-<foreign peptide>-50 e) 49-<foreign peptide>-51 f) 50-<heterologous peptide>-52 q) 72-<foreign peptide>-77 r) 73-<foreign peptide>-78 s)74-<foreign peptide>-79 t) 4-<foreign peptide>-5 The present invention relates to the above polypeptide, comprising:

[0061] More suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (d) to (f) and a second heterologous peptide insertion at any of positions (q) to (s).

[0062] More suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (d) to (f) and a second heterologous peptide insertion at position (t).

[0063] More suitably, the polypeptide comprises two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (q) to (s) and a second heterologous peptide insertion at position (t).

[0064] More suitably, the polypeptide comprises three heterologous peptide insertions: a first heterologous peptide insertion at any of positions (d) to (f), a second heterologous peptide insertion at any of positions (q) to (s), and a third heterologous peptide insertion at position (t).

[0065] Suitably, the polypeptide of the invention comprises an amino acid sequence corresponding to amino acids 1-98 of SEQ ID NO: 1, including the mutations described. A few of the mutations described are deletions, for example, the amino acid corresponding to amino acid 61 of SEQ ID NO: 1 may be mutated by substitution or by deletion. If mutated by deletion, the resulting final polypeptide will contain only 97 amino acids corresponding to SEQ ID NO: 1; in other words, if D61 is deleted, ΔD61 may correspond to D61 of SEQ ID NO: 1, and those skilled in the art will interpret such features accordingly. Most suitably, the mutations are substitutions.

[0066] In a broad aspect, a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence corresponding to amino acids 1 to 98 of SEQ ID NO: 1, For SEQ ID NO: 1, M65I, T51I, T51L, T51V, M65V, A59V, N32G, A59I, L38A, V20A, V20I, A40I, L38V, G50S, L38F, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34K, T34D, T34P, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, T51F, T51A, A59L, K63R, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D , Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N 62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) , (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) Also disclosed are polypeptides comprising a mutation selected from the group consisting of: and further comprising a heterologous peptide insertion. Suitably, the heterologous peptide insertion is a heterologous peptide inserted at one of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 Includes.

[0067] Affimers® are engineered non-antibody binding proteins (i.e., polypeptide affinity reagents). Affimer (AFFIMER) is a registered trademark.

[0068] As is known in the art, aptamers and Affimer® reagents are not the same. Affimer technology has been modified to overcome many of the problems associated with aptamers or antibodies and offers several advantages. For example, sensitivity to the assay environment is improved because Affimer® scaffolds tolerate a wide pH range, making them suitable for a wide range of assay conditions. Affimer® molecules also lack sensitivity to EDTA, a problem for aptamers that require Mg++ for folding and function. Aptamers are not constrained, whereas Affimer® proteins constrain the displayed heterologous peptide.

[0069] Suitably, Affimer® reagents, such as the Affimer® scaffolds / polypeptides described herein, are based on or derived from the human Stefin A protein, as described in more detail below.

[0070] Suitably the polypeptide is a Stefin A polypeptide.

[0071] In one aspect, the present invention relates to Stefin A polypeptides having a combination of one or more mutations from a set of defined mutations all sharing a common technical feature that will be demonstrated to affect stability, such as thermal stability (Tm), of the polypeptide and heterologous peptide insertion, and to the best of the inventors' knowledge or belief as of the filing date of this application, these combinations are novel.

[0072] A small number of amino acids at particular positions taught by the inventors may have previously existed in the art (e.g., as residues naturally occurring in less related Stefin A homologs, i.e., without a heterologous peptide insertion and therefore not present in the novel combination), or may have existed in combination with a heterologous peptide insertion, for example, in prior art scaffold STM. Examples of such mutations (substitutions) that may have previously existed in the art include V20A, T34K, L38F, Q42E, V48D, G50S, T51I, A59V, K63R, M65I, and N90T. In one embodiment, suitably, the polypeptide of the invention does not comprise a mutation selected from this group. Suitably, when the polypeptide of the invention has a mutation selected from this group, it does so in combination with at least one heterologous peptide insertion, most suitably at least one heterologous peptide insertion at one or more of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 It also has

[0073] Suitably, when a polypeptide of the invention has a mutation selected from this group, it also has at least one further mutation selected from Table A.

[0074] [Table 1]

[0075] Examples of such mutations (substitutions) that may have previously existed in the art for increasing the Tm of a Stefin A polypeptide include T34K, Q42E, G50S, T51I, A59V, K63R, M65I, and N90T. In one embodiment, suitably, a polypeptide of the invention does not comprise a mutation selected from this group. Suitably, when a polypeptide of the invention has a mutation selected from this group, it does so by incorporating at least one heterologous peptide insertion, most suitably at least one heterologous peptide insertion at one or more of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 It also has

[0076] Suitably, when a polypeptide of the invention has a mutation selected from this group, it also has at least one further mutation selected from Table B.

[0077] [Table 2]

[0078] Examples of such mutations (substitutions) that may have previously existed in the art for reducing the Tm of a Stefin A polypeptide include V20A, L38F, and V48D. In one embodiment, suitably, a polypeptide of the invention does not comprise a mutation selected from this group. Suitably, when a polypeptide of the invention has a mutation selected from this group, it does so by incorporating at least one heterologous peptide insertion, most suitably at least one heterologous peptide insertion at one or more of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 It also has

[0079] Suitably, when a polypeptide of the invention has a mutation selected from this group, it also has at least one further mutation selected from Table C.

[0080] [Table 3]

[0081] Suitably, the polypeptide comprises an amino acid sequence corresponding to amino acids 1 to 98 of SEQ ID NO: 1, including the mutations described above.

[0082] Thus, in one aspect, the invention provides a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence corresponding to amino acid residues 1-98 of SEQ ID NO: 1, the amino acid sequence has at least 80% identity to amino acid residues 1 to 11, 13 to 15, 17 to 19, 21 to 25, 27 to 28, 35 to 37, 39, 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53, 55 to 58, 63 to 64, 66, 68 to 82, 84 to 85, and 87 to 98 of SEQ ID NO: 1; The present invention relates to a polypeptide, characterized in that said polypeptide comprises one or more mutations selected from the group disclosed above relative to SEQ ID NO: 1.

[0083] Suitably, the polypeptide comprises at least 80 amino acids, suitably at least 81 amino acids, suitably at least 82 amino acids, suitably at least 83 amino acids, suitably at least 84 amino acids, suitably at least 85 amino acids, suitably at least 86 amino acids, suitably at least 87 amino acids, suitably at least 88 amino acids, suitably at least 89 amino acids, suitably at least 90 amino acids, suitably at least 91 amino acids, suitably at least 92 amino acids, suitably at least 93 amino acids, suitably at least 94 amino acids, suitably at least 95 amino acids, suitably at least 96 amino acids, more suitably at least 97 amino acids, and most suitably at least 98 amino acids (excluding any heterologous peptide insertions). Suitably, the polypeptide comprises an amino acid sequence corresponding to full-length hSteA. Suitably, full-length hSteA means 98 amino acids, most suitably corresponding to that set forth in SEQ ID NO:1.

[0084] scaffold As is well known in the art, the term "scaffold" refers to a protein that can present a target peptide to the solvent without its own structure being distorted by the target peptide.

[0085] Regarding the presentation of peptides to the solvent, this can be tested using immunoprecipitation experiments. For example, evidence that a peptide is presented to the solvent can be obtained by its availability with an antibody that can recognize it. Therefore, to test the ability of a scaffold protein to present a peptide to the solvent, a scaffold containing the peptide is expressed and an antibody that recognizes the peptide is used to immunoprecipitate the scaffold-peptide fusion. If the protein can be immunoprecipitated or captured with the antibody, this indicates that the peptide has been presented to the solvent as required by the scaffold protein. Another or alternative evidence that a peptide is presented to the solvent can be obtained by phosphorylation studies. By incorporating a phosphate acceptor site into the target peptide and then contacting the scaffold-peptide fusion with the cognate kinase under conditions that allow phosphorylation, the presentation of the peptide to the solvent can be verified. Peptide phosphorylation indicates proper presentation to the solvent.

[0086] The scaffold protein must be able to accept a peptide insert, i.e., a heterologous peptide insert ("target peptide") as taught herein. Preferably, the peptide insert is 36 amino acids or less, preferably 20 amino acids or less. Preferably, the peptide insert is 12 amino acids or less.

[0087] The resistance of a scaffold protein to deformation caused by a target peptide contained in the scaffold protein can be tested using techniques such as circular dichroism. Specifically, the circular dichroism analysis of a scaffold protein without a target peptide inserted should be substantially the same as the circular dichroism properties of the same scaffold protein when the target peptide is inserted. This similarity demonstrates that the presence of the target peptide in the scaffold protein has not damaged or distorted the structure of the scaffold protein containing the target peptide.

[0088] For example, near-UV CD spectra report on the environment of aromatic side chains. Positive or negative peaks in the near-UV CD spectrum indicate that the environment around the aromatic residue is fixed. This translates to a protein with a fixed tertiary structure. Incorporation of a heterologous peptide into an insertion site as taught herein suitably results in a near-UV CD spectrum with the same shape as an "empty" scaffold, indicating that the addition of the heterologous peptide did not disrupt the tertiary structure of the scaffold protein. As will be appreciated by those skilled in the art, the spectrum may have different magnitudes, e.g., higher peaks and / or lower troughs, but maintaining the same spectral shape is an important factor.

[0089] For example, far-UV CD spectra report on global secondary structure. Alpha helices, beta-strands, and random coils all have distinct far-UV CD spectra. Incorporation of a heterologous peptide into an insertion site as taught herein suitably results in a far-UV CD spectrum with the same shape as an "empty" scaffold, indicating that the addition of the heterologous peptide did not disrupt the global secondary structure of the scaffold protein. As will be appreciated by those skilled in the art, the spectrum may have different magnitudes, e.g., higher peaks and / or lower troughs, but retention of the same spectral shape is an important factor.

[0090] Suitably, scaffold protein constrains target peptide.The existence of the constraining effect of scaffold protein can be demonstrated by comparing the affinity of the entity that binds to target peptide when target peptide exists in scaffold protein with the affinity when peptide does not exist in scaffold protein.The difference between these two affinities indicates that scaffold protein constrains peptide to a specific three-dimensional conformation.Suitably, scaffold protein constrains peptide, and therefore when peptide exists in association with scaffold protein, demonstrates increased binding affinity.In other words, suitably, scaffold protein reduces the entropy consumption of binding, and therefore increases the measured affinity when compared with the binding of free peptide.

[0091] The scaffold protein is suitably biologically neutral. "Biologically neutral" means that known interactions with other proteins have been abolished. Furthermore, any signaling capabilities the protein may have are preferably removed. Mutations for biological neutrality of Stefin A-based scaffolds are known, and preferred examples are discussed herein. Combining such mutations for biological neutrality with the mutations taught herein for modulating thermostability is expressly contemplated.

[0092] Suitably, the polypeptide of the present invention is a scaffold protein.

[0093] In another aspect, the present invention relates to the use of a polypeptide as disclosed herein as a scaffold protein.

[0094] thermal stability An essential part of the present invention is the modulation of thermostability. Without knowledge of the present invention, in principle, mutation of any residue in the polypeptide of the present invention could result in a change in thermostability. However, the present inventors have studied the protein in detail at every level of its structure and designed specific changes to the protein to affect thermostability. Surprisingly, not all of the mutations designed by the present inventors necessarily produced the expected results. Thus, there is a great degree of unpredictability involved in making mutations to affect the thermostability of a scaffold protein. As a result of extensive intellectual effort and experimentation, the present inventors have arrived at the very detailed teachings presented herein regarding which residues can be used to affect thermostability.

[0095] Various data unexpectedly showing residues with no or neutral effect on stability such as Tm are also included for comparison, and these data further illustrate how predictions from the art could not have led to the present invention.

[0096] Thermostability can be assessed by any suitable means known in the art. Most suitably, thermostability is suitably read or assessed by measuring the Tm. Accordingly, reference to "thermal stability" can suitably be construed as a reference to "Tm." Unless otherwise clear from the context, "improved" thermostability / Tm means increased thermostability / raised Tm. "Increased" is a relative term, and unless otherwise clear from the context, "increased" means "increased / raised compared to wild-type Stefin A (in other words, compared to Stefin A having wild-type amino acid residues at defined positions, most suitably compared to wild-type Stefin A having the amino acid sequence of SEQ ID NO: 1)."

[0097] It should be noted that the majority of measurements presented herein are for hSteA Y35W, and its relative differences to hSteA are the scientific premise.

[0098] In addition to providing an assessment of thermal stability, Tm is a good proxy for assessing how easy it is to make a scaffold protein; scaffolds with higher Tm are advantageously easier to make. Furthermore, an increase in Tm can indicate that the protein is "rigider" and therefore more suitable for accepting heterologous peptide insertions. Furthermore, Tm is a good indicator of stability; for example, the shelf life of a protein can be advantageously increased if it is more stable, i.e., has a higher Tm. Furthermore, an increase in Tm can provide additional benefits, such as improved downstream formatting performance, long-term stability, and / or assay design flexibility.

[0099] The Tm of a protein, also known as the denaturation midpoint, is defined as the temperature at which both the folded and unfolded states are present in equal amounts or in equilibrium within a population. The Tm is thus determined assuming two-state protein folding.

[0100] The Tm values ​​given herein refer (unless otherwise indicated) to an "empty" polypeptide / scaffold protein of the invention, i.e., a scaffold protein without any heterologous peptide insertion. Insertion of heterologous sequences can, and often does, reduce thermal stability, such as Tm, and therefore the most meaningful values ​​are obtained when empty scaffold proteins are compared.

[0101] The melting temperature is a particularly useful indicator of protein stability. As used herein, melting temperature refers to the apparent midpoint of the thermal unfolding transition. The relative proportion of folded and unfolded protein can be determined by any technique known to those skilled in the art, including differential scanning calorimetry, UV difference spectroscopy, fluorescence, circular dichroism (CD), or NMR (see, e.g., Pace, C. Nick, and J. Martin Scholtz. "Measuring the conformational stability of a protein." Protein structure: A practical approach 2 (1997): 299-321).

[0102] Measurement method Several different measurement techniques can be used to assess the same property, such as Tm. Preferred techniques for measuring Tm include Optim (most suitably Optim 2) and / or differential scanning calorimetry (DSC). Most suitably, assessment of thermal stability, such as Tm, is carried out by differential scanning calorimetry (DSC) using any commercially available instrument and / or by using the "Optim 2" high-throughput protein stability measurement device (also known as UNit) available from Avacta Analytical (Unit 20, Ash Way Thorp Arch Estate, Wetherby LS23 7FA, UK) and / or from Unchained Labs (6870 Koll Center Parkway, Pleasanton, CA 94566, USA).

[0103] More specifically, any scientist / skilled person will understand that it is unlikely that a property will have the same absolute value when measured by two different techniques. That said, the Tm measured by Optim and by DSC will be very similar, though possibly not exactly identical, values. The Tm measured by DSC and the Tm measured by Optim will provide the same indication of change. Thus, if the Tm of a polypeptide is measured by Optim and the same peptide is measured by DSC, the measurements will not provide the exact same absolute value, but both will provide the same indication of change, e.g., +10°C. Furthermore, the ranking of individual polypeptides will be the same using either technique. Therefore, it should be kept in mind that absolute values ​​assigned according to normal scientific standards may vary slightly depending on the technique used to perform the measurement. Unless otherwise stated, values ​​herein are appropriately determined by Optim. Advantages of Optim include its speed and high reliability using very little input material. One limitation of Optim is that measurements are most reliable when the Tm is below 95°C, suitably below 92°C, and most suitably below 90°C. The advantage of DSC is that it works very well at higher temperatures and may be slightly better at avoiding interference effects. Therefore, if one wishes to attempt to determine an absolute Tm, DSC would be the measurement technique of choice. However, in practice, when measuring "delta" or differences between individual polypeptides, Optim is a very convenient, reliable, and robust technique to use. Unless otherwise stated, the values ​​provided herein are determined using Optim. In all circumstances, it is good scientific practice to compare measurements between individual polypeptides determined using the same measurement technique. Suitably, the Tm is the Tm determined by DSC or Optim. Most suitably, the Tm is the Tm when determined by Optim.

[0104] Most suitably, stability, such as Tm, is assessed for wild-type Stefin A in the same manner that stability, such as Tm, is assessed for polypeptides / scaffold proteins having mutations as disclosed herein. For reference purposes, the Tm of wild-type Stefin A, as assessed by DSC(r / t), is 89.0°C.

[0105] For comparison purposes, the prior art SQT scaffold protein has a Tm of 64.6°C when assessed by the same method.

[0106] Measurement techniques are discussed in more detail below.

[0107] Similarly, human SteA is a preferred starting point for the scaffold before mutating and / or inserting the described heterologous peptide. Advantageously, the Y35W mutation is included to provide a signal that can be measured to easily monitor thermal stability. Thus, many or most of the measurements provided were performed on scaffolds containing Y35W. For example, when we present data for N32G, which increases stability by 3 degrees, this measurement was that hSteA Y35W N32G increases stability by 3 degrees compared to hSteA Y35W.

[0108] It is a reasonable and robust scientific expectation that the effect of mutations as measured on hSteA Y35W will have the same effect on hSteA. This has been carefully verified. We present data to support this: we performed DSC experiments on multiple mutations in both hSteA and hSteA Y35W. We list these data below, thereby providing stability differences from hSteA / hSteA Y35W and demonstrating that this expectation is scientifically acceptable:

[0109] [Table 4]

[0110] Suitably, the polypeptides / scaffold proteins of the present invention have a melting temperature (Tm) of at least 90°C, more preferably at least 91°C, more preferably at least 92°C, more preferably at least 94°C, more preferably at least 95°C, more preferably at least 98°C, and most preferably at least 100°C.

[0111] In one aspect, the invention relates to increasing the thermostability of Stefin A polypeptides, such as Stefin A scaffold proteins, through the use of mutations as taught herein.

[0112] In another aspect, the present invention relates to reducing the thermal stability of a Stefin A polypeptide, such as a Stefin A scaffold protein, through the use of mutations as taught herein.

[0113] Thus, in one aspect, the present invention provides a method for increasing the thermostability of a Stefin A polypeptide, such as a Stefin A scaffold protein, by making one or more mutations in said Stefin A polypeptide, such as a Stefin A scaffold protein, wherein the mutations are: E29M, N32G, T34V, T34R, T34K, Q42E, T45I, T45V, T51F, T51V, T51L, T51I, A59L, A59I, A59V, K63R , M65V, M65I, L67I, N90T, (E29K, K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G6 0N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) and more suitably selected from the group consisting of: E29M, N32G, T34V, T34R, T45I, T45V, T51F, T51V, T51L, A59L, A59I, M65V, L67I, (E29K , K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G6 0N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) The present invention relates to a method for producing a pharmaceutical composition comprising the steps of:

[0114] Thus, in one aspect, the present invention provides a method for reducing the thermal stability of a Stefin A polypeptide, such as a Stefin A scaffold protein, by making one or more mutations in said Stefin A polypeptide, wherein the mutations are: A12I, A12V, I16L, V20A, V20I, V20L, Q26E, T31K, N32D, N32H, T34D, T34P, L38A, L38V , L38F, A40I, A40V, Q42D, V48E, V48D, V48G, V48A, V48L, T51A, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) and more suitably selected from the group consisting of: A12I, A12V, I16L, V20I, V20L, Q26E, T31K, N32D, N32H, T34D, T34P, L38A, L38V , A40I, A40V, Q42D, V48E, V48G, V48A, T51A, (V20I, L38A), (V20L, L38A), (V20 I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) The present invention relates to a method for producing a pharmaceutical composition comprising the steps of:

[0115] The present invention is advantageous in that the mutations taught herein are not interdependent (unless otherwise stated in the text). In other words, the mutations (or groups of mutations, if otherwise stated in the text) taught herein for affecting thermostability can be combined in various permutations to achieve the same technical benefit of the present invention, i.e., to increase (or decrease) thermostability. Thus, if a particular subset of mutations is desired for use in a peptide according to the present invention, a subset can be selected from the entire set of mutations disclosed herein. Advantageously, these mutations do not depend on other parts of the protein to produce their beneficial effect. Thus, if a large change in thermostability is desired, one skilled in the art would select a larger number of mutations such as those disclosed. If increased thermostability is desired, one skilled in the art can select mutations disclosed herein that increase thermostability, or any subset of these mutations combined to produce the desired level of increased thermostability.

[0116] Similarly, if reduced thermostability is desired, one skilled in the art can select mutations disclosed herein that reduce thermostability, or any subset of those mutations combined to provide the desired level of reduced thermostability.

[0117] Each of the mutations disclosed herein as useful for manipulating or modulating the thermal stability (e.g., Tm) of a polypeptide of the invention can be used independently. As explained, in some embodiments, a skilled operator may seek to select each of the mutations useful for increasing Tm to obtain a polypeptide with higher thermal stability (unless otherwise stated, higher / increased or lower / decreased is relative to the thermal stability of wild-type Stefin A, the sequence of which has been provided herein). In another set of embodiments, a skilled operator may select each of the disclosed mutations to lower or decrease the thermal stability (e.g., Tm) of a polypeptide, such as a scaffold protein, relative to wild-type Stefin A. In another set of embodiments, a skilled operator may select from mutations that increase thermal stability and from mutations that decrease thermal stability; such "mixed" embodiments advantageously allow a skilled operator to arrive at a more precise preselected thermal stability (e.g., Tm) for the polypeptide / scaffold protein thus produced. For example, if a particular thermal stability (e.g., Tm) is desired, then in selecting a particular mutant to use, one skilled in the art would make calculations to select mutations that are likely to increase thermal stability (e.g., toward the desired value). However, because each mutation will result in a slightly different increase in thermal stability (e.g., Tm), it may then be necessary to also select one or more mutations that decrease thermal stability to "balance" the overall desired selected thermal stability value. For example, if the desired thermal stability is 6° higher than wild-type Stefin A, it may be possible to arrive at a combined effect of increased thermal stability of 6° (+4, +4, -2) relative to wild-type Stefin A by selecting two different mutations that each increase thermal stability by 4° and one additional mutation that decreases thermal stability by 2°. Any such combination of mutations, clearly including combinations of mutations selected from both those that increase and those that decrease Tm relative to wild-type Stefin A, may be used by the skilled operator.This can be important when designing scaffolds that incorporate additional mutations, such as mutations useful for achieving biological neutrality. In particular, it may be desirable to use a V48 mutation, such as V48D, which inhibits domain-swap dimerization and is therefore clearly useful for demonstrating biological neutrality. However, this mutation also has a destabilizing effect on the polypeptide / scaffold protein (reducing thermal stability, such as Tm). Therefore, it becomes important to select mutations that "balance" the effect of selecting the V48D biologically neutral mutation to affect thermal stability and reach the desired value. An exemplary polypeptide / scaffold protein designed in this way might have the following mutations: V48D, N32G, M65I (referred to herein as 3T2).

[0118] Thus, the exemplary 3t series scaffolds are examples of "mixed" embodiments. In addition to the destabilizing V48D mutation, they have various numbers of stabilizing mutations to "restore" (i.e., return to wild-type Tm by raising the Tm from the destabilized, lower Tm V48D mutant) and further increase the Tm. Data demonstrating the Tm of various examples of such mixed embodiments are as follows: hSteA N32G V48D=3t1=85.6℃ hSteA N32G V48D M65I=3t2=89.2℃ hSteA N32G V48D M65I T51L=3t3=91.6℃ hSteA N32G V48D M65I Q42E=3t4=92.4℃ hSteA N32G V48D Q42E T51L M65I=3t5=94.7℃

[0119] Clearly, it will be possible to produce polypeptides / scaffold proteins according to the invention with a combination of mutations that result in some net change in thermostability, e.g., Tm, compared to wild-type Stefin A. For example, one such embodiment could include a polypeptide / scaffold protein having a V48D mutation that lowers Tm, and also having one or more mutations according to the invention that increase Tm, such that the final Tm of the polypeptide / scaffold protein may be higher or lower than the Tm of wild-type Stefin A, depending on the mutations selected. Suitably, therefore, the change in Tm compared to wild-type Stefin A is not critical, whether the scaffold protein is a scaffold protein of the invention (as defined in the claims); what is important is that the scaffold protein of the invention comprises at least one mutation as taught herein that affects Tm, e.g., to increase or decrease Tm compared to Stefin A. When these mutations are used in a polypeptide / scaffold protein in combination with other mutations, such as the known V48D mutation, the resulting polypeptide / scaffold protein also contains mutations disclosed herein to affect thermal stability, such as Tm, and is therefore considered part of the present invention, regardless of whether the final product has a higher or lower Tm than wild-type Stefin A.

[0120] The assessment of whether a mutation increases or decreases the Tm relative to wild-type Stefin A is suitably determined for each mutation (or, where clear from the context, a group of interdependent mutations, as appropriate) individually, i.e., when made individually (or, where clear from the context, as a single group of interdependent mutations, as appropriate) and when the polypeptide / scaffold protein so mutated is compared to wild-type Stefin A.

[0121] Nonetheless, in preferred embodiments of the invention, the polypeptide / scaffold protein has increased thermal stability compared to wild-type Stefin A. Thus, suitably, where a polypeptide / scaffold protein comprises a mutation (e.g., a V48D mutation) that reduces its thermal stability, such as Tm, compared to wild-type Stefin A, said polypeptide / scaffold protein suitably further comprises a mutation as disclosed herein sufficient to compensate for the decrease in thermal stability, such as Tm, caused by the mutation(s) (e.g., the V48D mutation), thereby still resulting in a polypeptide / scaffold protein with increased thermal stability compared to wild-type Stefin A.

[0122] Thus, suitably the polypeptide / scaffold protein has a Tm that is at least +1°C increased compared to wild type Stefin A, suitably at least +2°C increased, suitably at least +3°C increased, suitably at least +4°C increased, suitably at least +5°C increased, suitably at least +6°C increased, suitably at least +7°C increased, suitably at least +10°C increased, suitably at least +13°C increased, suitably at least +14°C increased, suitably at least +17°C increased compared to wild type Stefin A.

[0123] Examples of such values ​​for a particular mutation or combination of mutations include: hSteA N32G M65I +7.6℃ hSteA N32G M65I Q42E +10.0℃ hSteA N32G M65I Q42E T51L +14.4℃ hSteA N32G M65I Q42E T51L A59V dD61 +13.6℃ hSteA N32G M65I Q42E T51L A59V G60N dD61 N62G +17.1℃ Examples include:

[0124] Examples of mixed embodiments (i.e., sequences containing destabilizing mutations but with compensatory stabilizing mutations) can be made. As an example, scaffolds containing V48D are made and their Tm (values ​​shown below) are determined: hSteA N32G V48D M65I=3t2;Tm 89.2°C hSteA N32G V48D M65I T51L=3t3;Tm 91.6℃ hSteA N32G V48D M65I Q42E=3t4;Tm 92.4℃ hSteA N32G V48D Q42E T51L M65I=3t5;Tm 94.7℃ hSteA Y35W V48D N32G Q42E T51L M65I(A59V ΔD61)(E29K K30E E33K)=3r1;Tm 95.1℃ hSteA Y35W V48D N32G Q42E T51L M65I(A59V G60N ΔD61 N62G)(E29K K30E E33K)=3r2;Tm>98.3℃.

[0125] Exemplary scaffolds such as 3t1 may be slightly less stable than wild-type hSteA, and 3t2 is about as stable as hSteA (slightly more stable than hSteA), yet such scaffolds are still useful. For example, 3t1 has very few mutations (only three mutations) compared to wild-type, and therefore represents an excellent compromise between maintaining a sequence close to wild-type while still designing a useful scaffold with good stability. In the case of 3t1, the destabilizing V48D is strongly compensated for by two additional stabilizing mutations, resulting in a highly stable scaffold that still has the V48D mutation useful for biological neutrality.

[0126] Suitably, polypeptides of the invention include a D at position 48, eg V48D.

[0127] Reduced thermal stability Similarly, in certain peripheral embodiments of the invention, the polypeptide / scaffold protein has reduced thermal stability compared to wild-type Stefin A. Thus, suitably, where a polypeptide / scaffold protein comprises a mutation (e.g., an N32G mutation) that increases its thermal stability, such as Tm, compared to wild-type Stefin A, said polypeptide / scaffold protein suitably further comprises a mutation as disclosed herein sufficient to compensate for the increase in thermal stability, such as Tm, caused by the mutation(s) (e.g., the N32G mutation), thereby also resulting in a polypeptide / scaffold with reduced thermal stability compared to wild-type Stefin A.

[0128] For reference, wild-type human stefin A has a Tm of 89.0°C.

[0129] Thus, in some embodiments, suitably the polypeptide / scaffold protein has a Tm that is at least -1°C reduced compared to wild type Stefin A, suitably at least -2°C reduced, suitably at least -3°C reduced, suitably at least -4°C reduced, suitably at least -5°C reduced, suitably at least -6°C reduced, suitably at least -7°C reduced, suitably at least -8°C reduced, suitably at least -9°C reduced, suitably at least -10°C reduced, suitably at least -11°C reduced, suitably at least -12°C reduced, suitably at least -13°C reduced, suitably at least -15°C reduced, suitably at least -16°C reduced, suitably at least -21°C reduced compared to wild type Stefin A.

[0130] Examples of such values ​​for a particular mutation or combination of mutations include: hSteA Y35W V20I L38A -16.7℃ hSteA Y35W V20L L38A -15.3℃ hSteA Y35W Y54D T83D Q86E -21.1℃ Examples include:

[0131] A more appropriate example is hSteA N32D V48D Y54D T83D Q86E (Tm approximately 58°C) hSteA V48D Y35W V20A L38A (Tm approximately 57°C) hSteA V48D Y35W A40I T31K A12I V20I (Tm approx. 50℃) is.

[0132] Those skilled in the art will know that destabilizing mutations should be selected that simultaneously maintain the scaffold protein folding properly, for example, at room temperature, e.g., 18-24°C. Clearly, if all of the destabilizing mutations taught herein were simultaneously introduced into a SteA polypeptide, the peptide would likely have a Tm below room temperature, limiting its practical use. Thus, in this embodiment, suitably, mutations as taught herein are used in combinations that result in a polypeptide having a Tm of 25°C or higher, suitably 30°C or higher, suitably 40°C or higher, or suitably 50°C or higher. Thus, in this embodiment, suitably, mutations as taught herein are used in combinations that result in a polypeptide having a Tm of 25°C or higher, suitably 30°C or higher, suitably 40°C or higher, or suitably 50°C or higher. Thus, in this embodiment, suitably mutations as taught herein are used in combinations that result in a polypeptide having a Tm of 25-88°C or above, suitably 30-88°C or above, suitably 40-88°C or above, suitably 50-88°C or above.

[0133] Assessment of correct folding (e.g., structure or conformation) is taught herein, for example, using near- and far-UV CD spectroscopy. Thus, if in any doubt, one skilled in the art need only generate a protein with the combination of mutations to be checked and then assess correct folding (e.g., structure or conformation) and / or measure the Tm as taught herein.

[0134] The most preferred embodiments have increased thermostability compared to wild-type Stefin A.

[0135] P25 site P25 can be mutated. P25 mutations, such as P25S, can be useful to improve the peptide / scaffold protein, for example, by making it more rigid. The P25 site is located at a kink in the helix of stefin A-based scaffold proteins. Mutation of P25 to another residue (e.g., P25S) preserves the kink in the helix. P25 mutations, such as P25S, have no significant or no effect on thermal stability.

[0136] V48 part V48 is important for domain-swap dimerization. Suitably, V48 is mutated to prevent or inhibit domain-swap dimerization. Most suitably, V48 is V48D.

[0137] V48 is also important for cathepsin binding. Therefore, for better biological neutrality, V48 is suitably mutated. Most suitably, V48 is mutated to V48D.

[0138] The V48G or V48D mutation may be used, most suitably V48D.

[0139] N32 site Advantageously, the N32 site is mutated. For example, it can be mutated to N32G. This has the advantageous effect of increasing the thermal stability of the peptide / scaffold protein. In addition, this has the advantageous property of helping to ensure biological neutrality by removing the glycosylation site from the polypeptide / scaffold protein.

[0140] Other mutations at the N32 site may also be used. For example, an N32G mutation prevents glycosylation by removing the residue that would otherwise be glycosylated. However, N is only glycosylated if it is recognized in an NXS / T motif (where X is any residue except P). In the case of hSteA, the sequence at this site is NET. Glycosylation can be prevented either by mutating N to something else or by mutating T to something other than S. N32G has the added advantage of thermostability. Suitably, T34 substitutes any amino acid except S. T34E has the property of not altering thermostability. Either the N32 or T34 mutation will abolish glycosylation at N32 by removing the required motif.

[0141] N32G has an additional advantage in helix capping. G is found more than twice as frequently as average at this position. Histidine is the second most preferred, followed by N. All other mutations are less suitable than N for helix capping. Suitably, histidine at 32 (N32H) is not used because this amino acid residue adds a titratable side chain that may be undesirable.

[0142] An additional advantage of the N32G mutation is that it optimizes the cap at the end of the helix in this region of the polypeptide / scaffold protein, and therefore, although other N32 mutants may be used, N32G provides the specific advantages described above.

[0143] By convention, when certain mutations are bracketed together, for example, as shown in some figures or in tables herein, this indicates that the mutations are suitably made together, i.e., in combination. For example, "(T83D, Q86E)" means "a T83D substitution and a Q86E substitution" in the same polypeptide. In other words, mutations bracketed together are suitably made together in a polypeptide / scaffold protein according to the invention. This can be important, for example, when a pair of mutations is made in a "charge-swap" configuration. In this case, it is important to mutate one position to swap charges and make a corresponding opposite charge-swap mutation at the second site, thereby preventing charge-charge interactions between these two residues in the final polypeptide / scaffold protein. For example, the E29K K30E E33K mutations are surface charge mutations that are suitably made together and are therefore listed as a single option bracketed together: "(E29K K30 E33K)." That is, suitably, in an individual polypeptide / scaffold protein according to the invention, all three mutations E29K K30E E33K or none of the three mutations are made. The advantage of the (E29K K30E E33K) mutation set is the formation of favourable Coulombic interactions at the surface of the scaffold.

[0144] Reference sequence Suitably, all sequences herein are discussed with reference to human wild-type Stefin A (Cystatin A) having the sequence Uniprot P01040 (most suitably Uniprot P01040-1). For the avoidance of doubt, this sequence is provided below: SEQ ID NO: 1 - Uniprot P01040 - wild type human cystatin A

[0145] [ka]

[0146] When a particular amino acid residue is referred to herein using a numerical address, the numbering is done with reference to the wild-type stefin A (cystatin A) amino acid sequence (or with reference to the polynucleotide sequence encoding it, if a nucleic acid is referred to). An exemplary nucleic acid encoding wild-type stefin A (cystatin A) is: SEQ ID NO:2 (DNA sequence; artificial (not natural); amino acid sequence SEQ ID NO:1 was used to generate a codon-optimized DNA sequence suitable for expression in E. coli): ATGATTCCTGGTGGTTTGTCGGAAGCCAAACCGGCTACTCCGGAAATCCAGGAGATTGTGGACAAAGTCAAACCGCAACTGGAGGAAAAGACCAATGAAACCTATGGCAAACTCGAAGCGGTACAGTACAAAACCCAAGTCGTTGCG GGTACGAACTACTACATCAAAGTACGCGCAGGAGATAACAAGTATATGCATCTGAAAGTGTTCAAAAGCTTACCAGGGCAGAATGAGGATCTGGTTCTTACGGGCTATCAGGTGGATAAGAACAAAGACGATGAACTGACAGGCTTT

[0147] Where appropriate, reliance is placed on the latest version of the sequence database, or alternatively, on the release currently in effect as of the filing date of this application. For the avoidance of doubt, reliance is placed on UniProt Release 2017_02. More particularly, reliance is placed on UniProt Knowledgebase (UniProtKB) Release 2017_02, published February 15, 2017, by the UniProt consortium, European Bioinformatics Institute (EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource (PIR). UniProt (Universal Protein Resource) is a comprehensive catalog of information about proteins ("UniProt: the universal protein knowledgebase" Nucleic Acids Res. 45: D158-D169 (2017)).

[0148] This is used as is well understood in the art to locate the desired residue. This is not always a strict counting exercise, and attention must be paid to the context. For example, if the desired proteins are of slightly different lengths, locating the correct residue within the sequence may require the sequences to be aligned and the equivalent or corresponding residue to be selected. This is well within the purview of the skilled reader.

[0149] Mutating has its ordinary meaning in the art and may refer to the substitution, truncation, or deletion of one or more residues, motifs, or domains. Mutations may be made at the polypeptide level, for example, by synthesis of a polypeptide having a mutated sequence, or at the nucleotide level, for example, by creating a polynucleotide encoding the mutated sequence, which polynucleotide is then translated to produce the mutated polypeptide. Suitably, the mutations used are as described herein. Unless otherwise clear from the context, mutations referred to herein are substitutions. For example, "N32G" means that the residue corresponding to "N32" in wild-type Stefin A (SEQ ID NO: 1) is substituted with G.

[0150] Sequence variations The polypeptides described herein may contain sequence alterations compared to the wild-type sequence in addition to the key mutations described herein for modulating thermal stability, such as Tm. Specifically, the polypeptides described herein may contain additional sequence alterations at sites that do not significantly impair the function or behavior of the polypeptides described herein. The sequence alterations may be at the polypeptide level or at the nucleotide level.

[0151] Polypeptide function can be readily tested, for example, using methods such as those shown in the Examples section to verify that the peptide structure or conformation has not been significantly altered. Thus, sequence variations can be made in a polypeptide compared to a wild-type reference sequence, provided that the polypeptide retains its structure or conformation, which can be readily tested as shown herein.

[0152] For example, a polypeptide can be tested to determine whether it retains its structure or conformation by assessing its near-UV and far-UV CD spectra, recorded at 0.6 mg / mL and 0.2 mg / mL, respectively, in a compatible buffer (e.g., 50 mM sodium phosphate, pH 7.4). The spectra are processed by subtracting the buffer spectrum from the sample spectrum and converting units to molar ellipticity (for near-UV CD spectra) or mean residue ellipticity (for far-UV CD spectra). The near-UV CD spectrum should have a maximum between 275 nm and 280 nm, and an additional minimum near 295 nm if a single tryptophan is present at position 35. This could also be the maximum if a different position is selected for the tryptophan. The far-UV CD spectrum should resemble that of a typical beta-sheet protein, with a minimum near 218 nm. If these conditions are met, the polypeptide retains the correct structure or conformation. If there is no near-UV CD spectrum, or if the far-UV CD spectrum has minima at 208 nm and 222 nm, or a minimum at 198 nm, the polypeptide does not retain the correct structure or conformation and may be significantly altered.

[0153] Polypeptides include variants produced by introducing any type of additional alteration that can be deliberately modified (e.g., amino acid insertions, deletions, or substitutions; changes in glycosylation; changes affecting refolding or isomerization, three-dimensional structure, or self-association). Variants may have alterations that produce silent changes or that result in functionally equivalent polypeptides. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphipathic nature of the residues, provided that the structure or conformation of the polypeptide is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0154] Conservative substitutions can be made, for example, according to the following table: Amino acids in the same block in the second column and, suitably, in the same line in the third column may be substituted for each other:

[0155] [Table 5]

[0156] Preservation of the structure or conformation of the polypeptide is important when considering what mutations, substitutions, or other such changes can be made compared to the wild-type sequence. Typically, conservative amino acid substitutions will be less likely to adversely affect function.

[0157] When modifying a polypeptide to modulate thermostability as taught herein, the amino acid A12I, A12V, I16L, V20A, V20I, V20L, Q26E, E29M, T31K, N32G, N32D, N32H, T34V, T34R, T34K, T34D, T34P, L38A, L38V, L38F, A40I, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, G50S, T51F, T51V, T51L, T51I, T51A, A59L, A59I, A59V, K63R, M65V, M65I, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D , Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N 62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) , (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) should be substituted as taught herein, or, if unsubstituted, should appropriately correspond to SEQ ID NO: 1. Residues other than those shown in SEQ ID NO: 1, or substitutions other than those taught herein, appropriately are not used at these positions.

[0158] When modifying polypeptides to increase thermostability as taught herein, the amino acid E29M, N32G, T34V, T34R, T34K, Q42E, T45I, T45V, T51F, T51V, T51L, T51I, A59L, A59I, A59V, K63R , M65V, M65I, L67I, N90T, (E29K, K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G6 0N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) should be substituted as taught herein, or, if not substituted, should appropriately correspond to SEQ ID NO: 1. Residues other than those shown in SEQ ID NO: 1, or substitutions other than those taught herein, appropriately are not used at these positions.

[0159] When modifying a polypeptide to reduce its thermal stability as taught herein, the amino acid A12I, A12V, I16L, V20A, V20I, V20L, Q26E, T31K, N32D, N32H, T34D, T34P, L38A, L38V, L38F, A40I, A40V, Q42D, V48E, V48D, V48G, V48A, V48L, G50S, T51A, (V20I, L38A), (V20 L, L38A), (V20I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60 P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) should be substituted as taught herein, or, if not substituted, should appropriately correspond to SEQ ID NO: 1. Residues other than those shown in SEQ ID NO: 1, or substitutions other than those taught herein, appropriately are not used at these positions.

[0160] When modifying a polypeptide to provide aromatic residues for fluorescence / absorbance as taught herein, one or more of amino acids Y35W, Y43W, Y53W, Y54W, Y64W, F70W, or Y85W should be substituted as taught herein, or, if unsubstituted, appropriately correspond to SEQ ID NO: 1. Residues other than those shown in SEQ ID NO: 1, or substitutions other than those taught herein, suitably are not used at these positions.

[0161] Suitably, the polypeptides of the invention include a W at position 35, for example Y35W.

[0162] Note that Y85W shows a clear transition upon unfolding as judged by the intrinsic fluorescence emission spectrum of BCM and exhibits improved absorbance characteristics compared to hSteA, but does not show a clear improvement in the amplitude of the raw fluorescence emission spectrum. For the avoidance of doubt, Y85W can still be used to observe the transition upon unfolding, resulting in a small improvement. Thus, this mutation, while still useful for observing the transition, does not significantly increase fluorescence and is still functional in this setting.

[0163] When modifying a polypeptide for biological neutrality as taught herein, one or more amino acids from the table below should be substituted as taught herein, or, if not substituted, should appropriately correspond to SEQ ID NO: 1. Residues other than those shown in SEQ ID NO: 1, or substitutions other than those taught herein, suitably are not used at these positions:

[0164] [Table 6] TIFF0007659292000008.tif22161

[0165] Residues that are less conserved among proteins are more likely to tolerate mutations.

[0166] A total of up to 15 substitutions compared to SEQ ID NO:1 can be made to increase thermostability.

[0167] In addition, for biological neutrality, as many as two substitutions can be made compared to SEQ ID NO:1.

[0168] In addition, one substitution can be made compared to SEQ ID NO: 1 to provide an aromatic residue for fluorescence / absorbance.

[0169] Thus, in one embodiment, the present invention relates to polypeptides having up to 18 substitutions compared to SEQ ID NO:1.

[0170] By way of example, in one embodiment, the present invention relates to polypeptides having the following substitutions compared to SEQ ID NO: 1: One substitution for fluorescence (e.g., Y35W); two substitutions for biological neutrality (e.g., N32G, V48D); 15 substitutions for Tm (T34, Q42, T45, T51, K63, M65, L67, N90 (all single mutations), E29K K30E E33K (charge interaction), A59 G60 D61 N62 (b-turn)).

[0171] Additional mutations, most suitably substitutions, of one or more residues other than those explicitly mentioned above may be made, provided that the structure or conformation of the polypeptide is maintained. Thus, in one embodiment, the present invention relates to polypeptides having up to 25 mutations, most suitably substitutions, compared to SEQ ID NO: 1.

[0172] For example, (1 - novel and increased or decreased Tm):

[0173] [ka]

[0174] Wild type is A, mutant is I, V, X1 X2, where the wild type is I and the mutant is L Wild type is V, mutant is I, L, X3 Wild type is Q and mutant is E, X4 X5, where wild type is E and mutant is M X7, where the wild type is T and the mutant is K X8, where the wild type is N and the mutations are G, D, and H X10, where wild type is T and mutations are V, R, D, and P The wild type is L, and the mutants are A and V. X11 Wild type is A, mutant is I, V, X12 X13, where the wild type is Q and the mutant is D Wild type is T, mutant is I, V, X14 X15, where the wild type is V and the mutations are E, G, and A X16, where the wild type is T and the mutations are F, V, L, and A X18: Wild type is A, and mutants are L and I Wild type is D and mutant is Δ, X20 X22, where wild type is M and mutant is V X23, where the wild type is L and the mutant is I The wild type is (V, L) and the mutants are (I, A), (L, A), (I, V), (L, V), (X3, X11). The wild type is (E, K, E) and the mutant is (K, E, K), (X5, X6, X9) The wild type is (Y, T, Q) and the mutant is (D, D, E), (X17, X24, X25) The wild type is (A, G, D, N), and the mutants are (L, N, G, K), (L, N, Δ, G), (V, N, G, K), (I, N, G, K), (I, N, Δ, G), (V, N, Δ, G), (X18, X19, X20, X21). The wild type is (A, D, N) and the mutant is (V, N, K), (X18, X20, X21) The wild type is (A, D) and the mutant is (V, Δ), (X18, X20) The wild type is (G, D, N) and the mutations are (N, G, K), (N, Δ, G), (P, Δ, P), (P, P, K), (P, Δ, G), (P, G, K), (X19, X20, X21) The wild type is (T, Q) and the mutant is (D, E), (X24, X25) The wild type is (D, N) and the mutant is (N, K), (X20, X21)

[0175] Thus, in one embodiment, the present invention relates to polypeptides having up to 13 mutations, most suitably substitutions, compared to SEQ ID NO: 1, such as (2 - new and Tm-increasing):

[0176] [ka]

[0177] X1, where the wild type is E and the mutant is M X3, where wild type is N and mutant is G Wild type is T, mutant is V, R, X5 Wild type is T, mutant is I, V, X6 X7, where the wild type is T and the mutations are F, V, and L Wild type is A, and mutants are L and I, X8 Wild type is D and mutant is Δ, X10 X12, where wild type is M and mutant is V X13, where the wild type is L and the mutant is I The wild type is (E, K, E) and the mutant is (K, E, K), (X1, X2, X4) The wild type is (A, G, D, N), and the mutants are (L, N, G, K), (L, N, Δ, G), (V, N, G, K), (I, N, G, K), (I, N, Δ, G), (V, N, Δ, G), (X8, X9, X10, X11). The wild type is (A, D, N) and the mutant is (V, N, K), (X8, X10, X11) The wild type is (G, D, N), and the mutants are (N, G, K), (N, Δ, G), (X9, X10, X11). The wild type is (A, D) and the mutant is (V, Δ), (X8, X10)

[0178] Thus, in one embodiment, the present invention relates to polypeptides having up to 18 mutations, most suitably substitutions, compared to SEQ ID NO: 1, such as (3 - new and resulting in a reduced Tm):

[0179] [ka]

[0180] Wild type is A, mutant is I, V, X1 Wild type is I and mutant is L, X2 Wild type is V, mutant is I, L, X3 Wild type is Q and mutant is E, X4 X5, where the wild type is T and the mutant is K Wild type is N, mutants are D and H, X6 Wild type is T, mutants are D and P, X7 Wild type is L, mutant is A, V, X8 Wild type is A, mutants are I and V, X9 Wild type is Q and mutant is D, X10 X11, where the wild type is V and the mutations are E, G, and A X12, where the wild type is T and the mutant is A The wild type is (V, L) and the mutants are (I, A), (L, A), (I, V), (L, V), (X3, X8) The wild type is (Y, T, Q) and the mutant is (D, D, E), (X13, X17, X18) The wild type is (G, D, N), and the mutants are (P, Δ, P), (P, P, K), (P, Δ, G), (P, G, K), (X14, X15, X16). The wild type is (D, N) and the mutant is (N, K), (X15, X16) The wild type is (T, Q) and the mutant is (D, E), (X17, X18)

[0181] Thus, in one embodiment, the present invention provides a polypeptide having up to 28 mutations, most suitably substitutions, relative to SEQ ID NO: 1, suitably together with a heterologous peptide insertion, said heterologous peptide inserted at one of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 For example, (9 - those in which the Tm is increased or decreased):

[0182] [ka]

[0183] Wild type is A, mutant is I, V, X1 Wild type is I and mutant is D, X2 Wild type is V, and mutations are A, I, and L, X3 Wild type is Q and mutant is E, X4 X5, where wild type is E and mutant is M X7, where the wild type is T and the mutant is K X8, where the wild type is N and the mutations are G, D, and H Wild type is T and mutations are V, R, K, D, and P, X10 X11, where the wild type is L and the mutations are A, V, and F Wild type is A, mutant is I, V, X12 Wild type is Q, and mutants are E and D, X13 Wild type is T, mutant is I, V, X14 X15, where the wild type is V and the mutations are E, D, G, A, and L X16, where the wild type is G and the mutant is S X17, where the wild type is T and the mutations are F, V, L, I, and A X19, where the wild type is A and the mutations are L, I, and V Wild type is D and mutant is Δ, X21 Wild type is K and mutant is R, X23 Wild type is M, mutant is V, I, X24 X25, where the wild type is L and the mutant is I X28, where wild type is N and mutant is T The wild type is (V, L) and the mutants are (I, A), (L, A), (I, V), (L, V), (X3, X11). The wild type is (E, K, E) and the mutant is (K, E, K), (X5, X6, X9) The wild type is (Y, T, Q) and the mutant is (D, D, E), (X18, X26, X27) The wild type is (A, G, D, N), and the mutations are (L, N, G, K), (L, N, Δ, G), (V, N, G, K), (I, N, G, K), (I, N, Δ, G), (V, N, Δ, G), (X19, X20, X21, X22) The wild type is (A, D, N) and the mutant is (V, N, K), (X19, X21, X22) The wild type is (G, D, N), and the mutations are (N, G, K), (N, Δ, G), (P, Δ, P), (P, P, K), (P, Δ, G), (P, G, K), (X20, X21, X22) The wild type is (A, D) and the mutant is (V, Δ), (X19, X21) The wild type is (D, N) and the mutant is (N, K), (X21, X22) The wild type is (T, Q) and the mutant is (D, E), (X26, X27)

[0184] Suitably, when considering sequence identity, amino acids marked "X" are not considered; suitably, sequence identity is assessed across the amino acid residues designated or defined as above (i.e., the non-X residues).

[0185] If some further guidance is needed to assess sequence identity when the reference sequence has variable residues (e.g., "X") therein, sequence identity is calculated using the Protein BLAST server from the NIH (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The query sequence is the amino acid reference sequence with all Xs (Xn) removed. The subject sequence is the desired sequence to be compared to the query sequence with equivalent residues removed. The analysis is then performed, resulting in an Ident value.

[0186] for example, The sequence of hSteA containing X1 to X25 (reference sequence) is linked to the desired sequence, in this example canine SteA (cSteA): hSteA (SEQ ID NO: 1) MIPGGLSEAKPX1TPEX2QEIX3DKVKPX4LEX5X6X7X8X9X10YGKX11EX12VX13YKX14QVX15AGX16NYX17IKVRX18X19X20X21KYX22HX23KVFKSLPGQNEDLVLX24GYX25VDKNKDDELTGF cSteA (SEQ ID NO: 3) MMPGGLTEAKPA TPEV QEIA NEVKPQ LEE KTNET YQEF EA VE YKT QVV AGI NYY IKVRV GDN SYI HL KIFKGLPGQNPTLTLT GYQ TDKSKDDELTGF

[0187] All Xn are removed, as are the corresponding residues in the desired sequence cSteA: hSteA-query sequence MIPGGLSEAKPTPEQEIDKVKPLEYGKEVYKQVAGNYIKVRKYHKVFKSLPGQNEDLVLGYVDKNKDDELTGF cSteA-target sequence MMPGGLTEAKPTPEQEINEVKPLEYQEEVYKQVAGNYIKVRSYHKIFKGLPGQNPTLTLGYTDKSKDDELTGF

[0188] The query and subject sequences are compared using Protein BLAST. The results show that the sequences have 59 of 73 identical residues, resulting in 81% sequence identity.

[0189] Similarly, if a desired sequence is shorter than a reference sequence, e.g., SEQ ID NO: 1, with a "gap" or truncation, such gap or truncation will be assessed by sequence comparison and contribute a correspondingly lower sequence identity value. In this regard, sequence identity should be assessed over the entire length of SEQ ID NO: 1, as appropriate. By convention, a shorter sequence may be compared to a reference sequence (SEQ ID NO: 1) only over the length of the desired shorter sequence. However, unless otherwise clear from the context, discussion / determination of sequence identity (and sequence identity values ​​referred to herein) appropriately treats truncations as "gaps." Illustratively, if a desired sequence is only 49 amino acids long and is compared to SEQ ID NO: 1 (which is 98 amino acids long), the maximum sequence identity score would be 50% (any substitutions relative to SEQ ID NO: 1 would further reduce the sequence identity score). Thus, a sequence identity score of 50% for a desired polypeptide compared to SEQ ID NO:1 implies the presence of at least 49 amino acid residues in the desired polypeptide, which 49 are identical to their corresponding residues in SEQ ID NO:1.

[0190] Suitably, if a residue is not mutated as above, it is "wild type" as above, in other words, suitably, if a residue is not mutated as above, it is as shown in hSteA (SEQ ID NO: 1).

[0191] Sequence identity For accuracy, sequence relationships have been discussed in terms of substitutions relative to SEQ ID NO: 1 (wild-type human Stefin A), however, it may be desirable to consider sequence relationships in terms of sequence identity.

[0192] Sequence comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate the percent homology (e.g., percent identity) between two or more sequences.

[0193] The percent identity can be calculated over a contiguous sequence. That is, one sequence is aligned with the other sequence, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such an ungapped alignment is performed over only a relatively small number of residues (e.g., less than 50 contiguous amino acids).

[0194] While this is a very simple and consistent method, it fails to take into account, for example, that for an otherwise identical pair of sequences, a single insertion or deletion may result in subsequent amino acid residues not being able to align, thus resulting in a significant reduction in the percent homology (% identity) when a global alignment (alignment of the entire sequence) is performed. Therefore, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology (identity) score. This is achieved by inserting "gaps" during sequence alignment to maximize local homology / identity.

[0195] These more sophisticated methods assign a "gap penalty" to each gap present in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible (indicating a higher relatedness between the two compared sequences) will obtain a score higher than 1 with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost on the presence of a gap and a smaller penalty on each subsequent residue in the gap. This is the most commonly used gap scoring system. Higher gap penalties, of course, will produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified; however, it is preferred to use the default values ​​when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0196] Therefore, the calculation of maximum homology percentage first requires the generation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Other examples of software capable of performing sequence comparison include, but are not limited to, the BLAST package, FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410), and the GENEWORKS suite of comparison tools.

[0197] While the final percent homology can be measured in terms of identity, the alignment process itself is not typically based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​typically used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs typically use either the public default values ​​or a custom symbol comparison table, if provided. It is preferred to use the public default values ​​for the GCG package, or a default matrix such as BLOSUM62 for other software. Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0198] Suitably, the polypeptides of the invention have at least 80% sequence identity to SEQ ID NO:1, more suitably 85%, more suitably 88%, more suitably 90%, more suitably 92%, more suitably 94%, more suitably 95%, more suitably 96%, more suitably 97%, more suitably 98%, more suitably 99% identity to SEQ ID NO:1.

[0199] In one embodiment, more suitably, the polypeptide of the present invention has at least 80% sequence identity, more suitably 85%, more suitably 88%, more suitably 90%, more suitably 92%, more suitably 94%, more suitably 95%, more suitably 96%, more suitably 97%, more suitably 98%, more suitably 99% identity to those residues of SEQ ID NO: 1 other than those specifically set forth as substituted in the definition of the polypeptide of the present invention under consideration (e.g., in the appended claims). In other words, in this embodiment, more suitably, the percent sequence identity is assessed for the desired sequence compared to SEQ ID NO: 1 excluding those residues specifically defined (e.g., substituted) in the particular embodiment of the polypeptide under consideration.

[0200] Suitably, any heterologous peptide insertions are excluded from the percent identity calculation.

[0201] In one embodiment, suitably, substitutions and any heterologous peptide insertions are as defined and sequence identity is judged against the remaining "background" or "backbone" sequence of the polypeptide compared to the corresponding residues of SEQ ID NO:1.

[0202] In all discussions of sequence identity, it should be noted that SEQ ID NO: 1 is 98 amino acids long. Thus, each single substitution corresponds to a 1.020408% change in identity when all 98 amino acids are considered. The above values ​​are given in integer percentage points and should be interpreted accordingly, taking into account that some amino acids within a polypeptide sequence cannot be substituted. Obviously, when fewer than 98 amino acids are considered (e.g., when considering sequence identity to residues of SEQ ID NO: 1 other than those specifically stated to be substituted in the definition of the polypeptide of the present invention being considered), each single amino acid substitution may correspond to a greater than 1.020408% change in identity. The skilled reader will be able to interpret the values ​​accordingly, taking into account that some amino acids within a polypeptide sequence cannot be substituted.

[0203] If any further guidance is needed, the table below shows how the percent sequence identity value changes when taking into account the number of residues compared and the number of amino acid substitutions made within that number of residues compared. Any further values ​​beyond the examples in the table can be readily calculated by the skilled researcher.

[0204] [Table 7]

[0205] The skilled reader will be aware that when an amino acid sequence identity value is given for a particular polypeptide, that particular polypeptide may also include a heterologous peptide insertion, and that this must be borne in mind when assessing sequence identity. Suitably, sequence identity is assessed over the amino acid residues corresponding to those in wild-type Stefin A of SEQ ID NO: 1. In other words, suitably, the assessment of sequence identity is considered over the amino acids corresponding to those present in SEQ ID NO: 1, but does not include the heterologous peptide insertion (or lack thereof).

[0206] In one embodiment, more suitably, sequence identity is considered over the amino acids corresponding to those present in SEQ ID NO: 1, but excluding any heterologous peptide insertions and without taking into account any substitutions already defined in the claims.

[0207] Most suitably, percent sequence identity is calculated on amino acid residues excluding those substituted for reasons of thermostability.

[0208] In some applications, for example, in contemplated therapeutic applications, it is desirable to minimize the number of mutations in the polypeptide / scaffold protein used. Thus, in some embodiments, suitably, the polypeptide / scaffold protein comprises five or fewer, suitably four or fewer, suitably three or fewer, suitably two or fewer mutations compared to wild-type human Stefin A. In this context, "mutation" refers to a point mutation, or the insertion or deletion of a small number of amino acids, for example five or fewer. The insertion of heterologous peptides into the polypeptide / scaffold protein of the invention is discussed separately. The insertion of one or more heterologous peptides is not considered a "mutation" in this context. Most suitably, mutation refers to a substitution.

[0209] It is noted that the standard in the art is the SQT scaffold protein as disclosed in WO 2009 / 136182, which has low thermal stability / Tm of 64.6°C. Suitably, the SQT scaffold is a scaffold having the sequence (SQT) of SEQ ID NO: 24 of WO 2009 / 136182: MIPRGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLAS TNYYIKVRAG DNKYMHLKVF NGPPGQNADR VLTGYQVDKN KDDELTGF.

[0210] Thus, embodiments of the present invention exhibit significant advantages over prior art SQT scaffolds. However, it should be noted that the comparative values ​​presented herein are relative to wild-type Stefin A, unless otherwise apparent from the context. Thus, it is possible to have a polypeptide / scaffold protein as disclosed herein that has a reduced (lower) Tm relative to wild-type Stefin A, but nevertheless provides the advantage of an improved (higher) Tm relative to SQT. While all values ​​are expressed relative to wild-type Stefin A for ease of understanding and consistency (unless otherwise apparent from the context), the advantages of the present invention can be obtained by modulating the Tm, regardless of whether the resulting Tm is higher or lower than that of wild-type Stefin A. The Tm desired by an operator of the present invention can be higher or lower than that of wild-type Stefin A or SQT. The key advantage is that the skilled reader can now alter / modulate the Tm of a polypeptide / scaffold protein as taught herein.

[0211] Suitably, the scaffolds of the invention have a Tm higher than SQT, ie higher than 64.6° C. Most suitably, the scaffolds of the invention have a Tm higher than wild type Stefin A, ie higher than 89.0° C.

[0212] It is an advantage of the present invention that a polypeptide / scaffold protein is provided that is biologically neutral.

[0213] It is an advantage of the present invention that a polypeptide / scaffold protein is provided that lacks glycosylation sites.

[0214] It is an advantage of the present invention that polypeptides / scaffold proteins are provided that have reduced or improved tendency to dimerize. This is advantageous when the scaffold is a drug, because dimerization can alter pharmacokinetics. This is also advantageous because dimerization can affect affinity and therefore interaction with the target. This is also advantageous because dimerization can affect immunogenicity. Dimerization can also change the effect of certain molecules from agonist to antagonist (or vice versa). Additionally, in some cases, dimerization can promote higher-order structure formation and destabilization over time. Furthermore, having monomers (i.e., reducing dimerization) has certain advantages. For example, monomers are smaller (e.g., improving tumor penetration).

[0215] Clearly, if a dimer is desired by the skilled operator, the dimerization residues can be treated accordingly, e.g., retained as wild type, such as V48, to allow / facilitate domain-swap dimerization if this is deemed advantageous for a particular application.

[0216] Heterologous peptide insertion Suitably, the heterologous peptide comprises 36 or fewer amino acids, more suitably 20 or fewer amino acids, more suitably 12 or fewer amino acids.

[0217] Suitably, the heterologous peptide comprises 3 or 4 or more amino acids, more suitably 12 or 13 or more amino acids, more suitably 20 or 21 or more amino acids, more suitably up to 36 amino acids.

[0218] Suitably, the heterologous peptide comprises 3 to 36 amino acids, more suitably 3 to 20 amino acids, more suitably 3 to 12 amino acids, more suitably 3 to 9 amino acids, more suitably 6 to 36 amino acids, more suitably 6 to 20 amino acids, more suitably 6 to 12 amino acids, more suitably 6 to 9 amino acids, and most suitably 9 amino acids.

[0219] It is an advantage of the present invention that a small increase in thermostability is observed when a heterologous peptide insert is included in the polypeptides of the present invention as taught herein.

[0220] Position of heterologous peptide insertion A heterologous peptide sequence can be inserted between amino acids 46 and 54, inclusive (e.g., as described in WO 2006 / 131749 (depicting scaffolds such as "STM") or, e.g., as described in WO 2009 / 136182 (depicting scaffolds such as "SQT")), more suitably between amino acids 46 and 50, inclusive, and more suitably between amino acids 48 and 50, inclusive. "Between ... inclusive" in this context means that the recited amino acids can be deleted in favor of the heterologous peptide insertion; i.e., any or all of amino acids 46-54 (for example) can be deleted and the heterologous peptide insertion can be made in their place. Thus, a deletion of amino acids 46-54 and an insertion to replace them is encompassed by the phrase "between amino acids 46 and 54." Amino acids 46 and 54 need not be retained. That is, rather than requiring the actual presence of amino acid 46 and amino acid 54 to be "between" them, the peptide is inserted between these positions with respect to SEQ ID NO: 1. If all of amino acids 46-54 are deleted, this can be represented as "45-<heterologous peptide>-55."

[0221] A heterologous peptide sequence can be inserted between amino acids 67-84, inclusive. A heterologous peptide sequence can be inserted between amino acids 71-73, inclusive. A heterologous peptide sequence can be inserted between amino acids 82-83, inclusive. The "Leu 73" site is an insertion site for a heterologous peptide (sometimes referred to as a "target peptide"). This represents a solvent-exposed loop of the Stefin A protein and is therefore applicable to presenting a target peptide in a solvent-accessible manner. The term "Leu 73 insertion" is used herein to describe the insertion of a heterologous peptide near, or appropriately into (e.g., within) the L73-L80 loop of human Stefin A. This term can refer to the addition of, insertion at, or replacement of Leu 80 of human Stefin A. More appropriately, this term refers to the addition of, insertion at, or replacement of Leu 73 of human Stefin A. In one embodiment, the Leu73 mutation can include replacing the entire loop between L73 and L80 with a heterologous peptide sequence. The heterologous peptide sequence can be inserted between amino acids 73 and 80, inclusive.

[0222] For example, the insertion can be done like this (using an SQT array as an example):

[0223] [ka]

[0224] Thus, suitably, loop 2 has a heterologous peptide sequence inserted between residues 49 and 50 (49-<heterologous peptide>-50), and loop 4 has a heterologous peptide sequence inserted between residues 73 and 78 (73-<heterologous peptide>-78) if residues 74 to 77 are replaced.

[0225] A heterologous peptide sequence can be inserted at the G4 site. A heterologous peptide sequence can be inserted at position 4. A heterologous peptide sequence can be inserted adjacent to position 4, i.e., between position 4 and the center of the polypeptide, for example, between amino acids 4 and 45, inclusive, or between amino acids 4 and 44, inclusive. Suitably, the G4 residue is retained. Most suitably, the G4 residue is mutated to G4R. Thus, suitably, a heterologous peptide sequence can be inserted between amino acids 5 and 45, inclusive, or between amino acids 5 and 44, inclusive. More suitably, a heterologous peptide sequence can be inserted between amino acids 4 and 5, inclusive, most suitably immediately after amino acid 4, e.g., at position 5.

[0226] The heterologous peptide can be inserted into one of the following positions in the protein (see, for example, WO 2014 / 125290 (depicting scaffolds such as "plant adhyron")): the loop between the first and second strands of the beta-sheet (sometimes referred to as loop 1); and / or the loop between the third and fourth strands of the beta-sheet (sometimes referred to as loop 2). In this context, first, second, third, and fourth refer to the protein sequence, i.e., from the N-terminus to the C-terminus of the protein. Suitably, the heterologous peptide is inserted into both of these positions, i.e., loop 1 and loop 2.

[0227] Loop Nomenclature The secondary structure of polypeptides / scaffold proteins can be modeled using open source software, most suitably "PPopen" available from the Technical University of Munich, Germany. Numbering of structures such as loops in the polypeptides / scaffold proteins of the invention follows the systematic nomenclature obtained using the PPopen software.

[0228] It should be noted that prior art documents used informal nomenclature. Thus, disclosures in prior art documents such as WO 2006 / 131749 and / or WO 2009 / 136182 and / or WO 2014 / 125290 may have referred to the informal name "Loop 1", which in fact refers to "Loop 2" when using the systematic nomenclature according to the PPopen software. The table below shows the nomenclature for the various structural parts of proteins, which will be followed throughout the text unless otherwise indicated by the text.

[0229] Unless otherwise clear from the context, the following loop nomenclature will be followed: Amino acid residue numbers are as in or correspond to SEQ ID NO:1.

[0230] [Table 8]

[0231] As used herein, the term "loop" means a "bound canonical secondary structure" (ie, alpha / beta).

[0232] Sometimes informal nomenclature is used in connection with particular embodiments of the invention. For example, preferred scaffolds are sometimes referred to as "3T" or "3R." Specific configurations of preferred embodiments of the invention are always disclosed with specific reference to the mutated residues as indicated in the text.

[0233] Suitably, a population or library of scaffold polypeptides is generated, each having a different heterologous peptide sequence (most suitably one per scaffold polypeptide).

[0234] "Heterologous" has its ordinary meaning, i.e., the inserted polypeptide has an amino acid sequence that is heterologous to the sequence derived from or corresponding to the Stefin A sequence being used, e.g., SEQ ID NO: 1. Heterologous may therefore also mean from another species and / or from a polypeptide other than human wild-type Stefin A (SEQ ID NO: 1). Most suitably, the heterologous polypeptide insertion comprises an artificial amino acid sequence, most suitably an artificial amino acid sequence designed by an operator.

[0235] A test heterologous peptide insertion can be used, for example, a GGS repeat, such as a nonamer peptide containing GGSGGSGGS. This is a particularly demanding peptide because it is entropically very difficult to access. Therefore, by inserting this as a heterologous peptide into the polypeptide / scaffold protein of the present invention, it is demonstrated that the scaffold performs very well in presenting such a soluble and entropically difficult to access loop. The ability to present such a loop is a very strong indicator of the stability of the polypeptide / scaffold protein of the present invention.

[0236] Particularly suitable sites for inserting heterologous polypeptides It should be noted that the most preferred sites for heterologous polypeptide insertion in the polypeptides / scaffold proteins of the present invention are themselves different from the sites disclosed for insertion in the prior art disclosures of Stefin A-based polypeptides / scaffold proteins.

[0237] Single Insert - Loop 2 48-<foreign peptide>-50 49-<foreign peptide>-51 50-<foreign peptide>-52

[0238] Single Insert - Loop 4 72-<foreign peptide>-77 73-<foreign peptide>-78 74-<foreign peptide>-79

[0239] For the avoidance of doubt, "48-<heterologous peptide>-50" means between the amino acid corresponding to position 48 of SEQ ID NO: 1 and the amino acid corresponding to position 50 of SEQ ID NO: 1, etc. Similarly, "72-<heterologous peptide>-77" means between the amino acid corresponding to position 72 of SEQ ID NO: 1 and the amino acid corresponding to position 77 of SEQ ID NO: 1, with the intervening amino acid deleted (i.e., replaced by a heterologous peptide insertion)), etc.

[0240] Combination - Double Insertion A combination of two insertions, one into loop 2 and one into loop 4, can be made as disclosed in FIG.

[0241] Further combinations In addition to the specific insertion examples described above, single or double insertions can be combined with additional insertions at the N-terminus of the protein, for example, proximal to the G4 site as disclosed above, thereby making double insertions (G4 site + Loop 2 or G4 site + Loop 4) or triple insertions (G4 site + Loop 2 + Loop 4).

[0242] Most suitably, the heterologous peptide is inserted between residues D48 and G50 with residue A49 deleted (referred to as "48-<heterologous peptide>-50").

[0243] Most suitably, the heterologous peptide is inserted between residues L73 and E78 (termed "73-<heterologous peptide>-78"), with residues P74, G75, Q76 and N77 deleted.

[0244] Most suitably, a double insertion is made by inserting a first heterologous peptide at 48-<first heterologous peptide>-50 and introducing a second heterologous peptide at 73-<second heterologous peptide>-78.

[0245] The first and second heterologous peptides are suitably different from each other.

[0246] The first, second and third heterologous peptides are suitably different from each other.

[0247] Exemplary sequences showing exemplary insertions are as follows (X = any amino acid; in these examples, the heterologous peptide insertion is 9 amino acids in length): Exemplary Scaffold 3r2 MIPGGLSEAKPATPEIQEIVDKVKPQLEKETGKTWGKLEAVEYKTQVD48XXXXXXXXXG50LNYYIKVRVN-GKYIHLKVFKSL73XXXXXXXXXE78DLVLTGYQVDKNKDDELTGF Exemplary scaffolding 3t4 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVEYKTQVD48XXXXXXXXXG50TNYYIKVRAGDNKYIHLKVFKSL73XXXXXXXXXE78DLVLTGYQVDKNKDDELTGF

[0248] The particular preferred insertion points taught herein provide certain additional advantages. These advantages result from the selection of the insertion site and may therefore be advantageously used for polypeptides / scaffold proteins having mutations different from those disclosed herein. In other words, the disclosure of particular insertion sites made in this document is a separate disclosure of the advantageous properties provided by the technical features. Thus, in one aspect, the present invention may relate to polypeptides / scaffold proteins, such as Stefin A-based polypeptides / scaffold proteins, having heterologous peptide(s) inserted at specific position(s) as disclosed herein. For example, single insertions - Loop 2: 48-<heterologous peptide>-50; 49-<heterologous peptide>-51; 50-<heterologous peptide>-52; single insertions - Loop 4 - 72-<heterologous peptide>-77; 73-<heterologous peptide>-78; 74-<heterologous peptide>-79; combinations - double insertions - a combination of two insertions, one inserted into Loop 2 and one inserted into Loop 4, can be made as disclosed in Figure 1.

[0249] affinity maturation Affinity maturation may be carried out by any suitable method known in the art, for example, one heterologous peptide from a desired binder may be held constant while only the second or further heterologous peptide insertion is varied to construct a "mini-library."

[0250] Another approach is alanine scanning of heterologous peptide inserts to identify the amino acids most important for interaction with the target.

[0251] An alternative approach is to perform "slippy PCR" and rescreen the resulting variants for heterologous peptide insertions against the target.

[0252] It is understood that in wild-type stefin A, one of its binding partners (a cathepsin) binds via all three loops to the relevant surface of the stefin A molecule. Therefore, another approach to affinity maturation is the introduction of a heterologous peptide into the third position at the N-terminus of the protein (the region containing the G4 residue of stefin A) and rescreening the scaffold protein bearing that heterologous peptide set against the target.

[0253] As used herein, "d" means "deletion of" (e.g., "dD61" means "deletion of D61"), which, as is common in the art, is usually designated as "ΔD61" (i.e., delta D61). Residue numbering is always according to human wild-type SteA, i.e., SEQ ID NO: 1.

[0254] In other words, when a deletion mutation is made in a polypeptide / scaffold protein according to the invention, the numbering still follows that of the human wild-type Stefin A protein. For example, if amino acid 61 is deleted, the numbering of that particular embodiment of a polypeptide / scaffold protein of the invention will jump from amino acid 60 directly to amino acid 62. In other words, the amino acid numbering is maintained relative to the human wild-type Stefin A reference sequence, rather than being adjusted to assign consecutive numbers to each of the amino acids present in a particularly preferred embodiment of a polypeptide / scaffold protein of the invention. This is conventional and readily understood by those skilled in the art as it is commonplace in the field of molecular biology.

[0255] It should be noted that multiple insertion sites for heterologous peptides are disclosed. Thus, a polypeptide / scaffold protein may contain a single heterologous peptide insertion, two heterologous peptide insertions at two different insertion sites (i.e., a single insertion at the first and second sites for a total of two heterologous peptide insertions), or three heterologous peptide insertions at three different insertion sites (i.e., a single insertion at the first, second, and third sites for a total of three heterologous peptide insertions). A library comprising polypeptides / scaffold proteins may contain a single heterologous peptide insertion per polypeptide / scaffold protein, two heterologous peptide insertions per polypeptide / scaffold protein, or three heterologous peptide insertions per polypeptide / scaffold protein.

[0256] Embodiments of the present invention that result in reduced thermal stability find application in situations where it is desirable to remove the functionality of a polypeptide / scaffold protein by heat treatment. One example is reducing the Tm of Affimer® proteins, which are designed to inhibit enzymes, such as enzymes that are enabled to function by heating to a set temperature (e.g., hot-start polymerases). For example, Affimer® reagents, such as polypeptide / scaffold proteins containing heterologous peptide inserts described herein, can be used to inhibit thermostable nucleic acid polymerases. These find application in PCR reaction mixtures. For example, it can be useful to produce Affimer® reagents, such as polypeptide / scaffold proteins according to the present invention, that inhibit the exonuclease function of a thermostable nucleic acid polymerase. This is particularly useful when primers are premixed into a PCR reaction mixture and then stored until use. If the exonuclease function of the thermostable nucleic acid polymerase is not inhibited, it may degrade the primers in the reaction mixture. However, this drawback of prior art reaction mixtures can be circumvented by including in the reaction mixture a polypeptide / scaffold protein according to the present invention that is capable of inhibiting the exonuclease function of the polymerase. What is important in this scenario is that the polypeptide / scaffold protein can be rendered inactive (e.g., denatured) to allow the PCR reaction to proceed.

[0257] Suitably, therefore, the polypeptides / scaffold proteins used in this application have reduced thermostability and are therefore degraded (e.g. denatured) during the first thermal cycle of the PCR reaction, thereby removing inhibition of the exonuclease function of the thermostable polymerase and allowing the reaction to proceed.

[0258] Another example is an affinity ligand that can co-elute with the target protein but can then be removed (or its function removed) by a heating step, in which case the Affimer® protein is designed to have a lower Tm than the target protein.

[0259] fluorescence It can be useful to replace aromatic amino acids in proteins, such as phenylalanine, with alternative aromatic amino acids that exhibit fluorescence and / or absorbance at 280 nm properties. For example, phenylalanine can be mutated to tyrosine (which has weak fluorescence properties) or to tryptophan (which has strong fluorescence properties). There are eight sites in wild-type Stefin A where these mutations are possible. We have tested these. The mutations and their utility in these applications are shown in the table below.

[0260] [Table 9]

[0261] From this, it can be concluded that some of the above mutations are useful for determining absorbance at 280 nm, for example, in determining protein concentration. Furthermore, certain mutations may be useful for introducing fluorescence, which is useful for monitoring protein conformational changes, such as protein folding.

[0262] More suitably, the polypeptide of the invention comprises a substitution relative to SEQ ID NO: 1 selected from the group consisting of Y35W, Y43W, Y53W, Y54W, Y64W, F70W and Y85W, all of which have good fluorescent properties.

[0263] Suitably, the polypeptides of the invention comprise a substitution selected from the group consisting of Y35W, Y43W and Y53W relative to SEQ ID NO: 1. These have the best fluorescent properties.

[0264] According to the above table, the most preferred mutation in a polypeptide / scaffold protein according to the present invention would be Y35W, which has the advantage of being the least destabilizing of the mutations and also provides the best spectrum for analysis.

[0265] It should be noted that each tryptophan introduced into a polypeptide / scaffold protein of the invention will destabilize it to some degree. This may be advantageous when considering embodiments of the invention that result in reduced thermal stability. Alternatively, the reduced stabilizing effect of these mutations can be ameliorated by making other mutations to increase thermal stability, as taught in more detail throughout this application. Thus, in some embodiments, a polypeptide / scaffold protein of the invention may contain both stabilizing and destabilizing mutations, depending on the purpose or properties of that particular polypeptide / scaffold protein.

[0266] The N32G mutation is advantageous as it results in a particularly strong increase in thermostability, eg, an increase of approximately 3°C.

[0267] The advantage of the preferred M65I mutation is that it results in a particularly strong increase in thermostability of +6°C.

[0268] It is noted that each mutation described herein as increasing the thermostability of the polypeptides / scaffold proteins of the invention is, in itself, novel.

[0269] It should be noted that cystatin C is only loosely related in sequence to cystatin A (stefin A). Cystatin C is naturally a dimer and, compared to stefin A, is a longer protein with a lower level of sequence identity. Although the cystatin fold is similar between cystatin A and cystatin C, these proteins share only one region of homology, corresponding to amino acids 49-95 of cystatin C, which correspond to amino acids 16-60 of cystatin A. Over this related region, only 13 / 47 amino acids are identical, corresponding to 28% sequence identity.

[0270] Subset of mutations Mutations can be selected from a group to increase the packing of the hydrophobic core.

[0271] Mutations can be selected from a panel to increase charge interactions on the surface of the polypeptide / scaffold protein.

[0272] Mutations can be selected from a group to increase the geometry of a particular turn in the polypeptide structure. By "increase," we mean making the turn energetically more favorable, e.g., energetically more stable. An example of one such mutation is the deletion of D61 (ΔD61).

[0273] Mutations can be selected from a panel to increase stability while still operating within the constraints of three-dimensional structure, for example, at the end of a β-turn. More specifically, mutations can be carefully selected to preserve the I-I+4 hydrogen bond configuration at the end of a particular β-turn.

[0274] Mutations can be selected from a group to improve stability and also advantageously change the type of turn within the three-dimensional structure of the protein, for example, mutations can be made to change a β-turn from type 1 to type 2 if desired.

[0275] It should be noted that mutations useful for achieving the disclosed technical features and advantages are taught generically in this application. For example, with respect to a mutation at position Q42, an E to D mutation is broadly considered a conservative substitution because it effectively removes only a single methylene group from the amino acid side chain. However, as can be seen from the data contained in this application, a Q42E mutation results in an increase in thermal stability of +3°C, while a Q42D mutation results in a decrease in thermal stability of -3°C. Thus, a seemingly conservative substitution that results in a seemingly very small change in the structure of the amino acid residue at this position can result in a dramatic difference of 6°C between the Tm of two polypeptide / scaffold protein variants. Therefore, the data in this application support the unity of the invention among each of the various mutations disclosed, which were carefully selected, designed, tested, and demonstrated to each achieve the same single specific technical effect. Furthermore, the claims of this application are limited only to those variants that are found and demonstrated to have and achieve that known specific technical effect. Thus, the various variations set forth in the appended claims indeed relate to only a single invention and satisfy the requirement of unity.

[0276] In all cases, references to "increased" or "decreased" stability, as reflected, for example, by an increase or decrease in the Tm of a particular polypeptide / scaffold protein, are quoted relative to the properties of human stefin A, i.e., the wild-type human stefin A polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1. Similarly, all mutations are described with reference to the same wild-type stefin A sequence, SEQ ID NO: 1.

[0277] The following groups of mutations are particularly advantageous for the following reasons:

[0278] Solvent exposed type Mutations at these positions improve the charge network on the surface of the protein, which helps make the protein more stable. Charge-altering amino acid substitutions can be made more easily on the surface of the Stefin A protein, which is advantageous. In addition, mutating residues on the protein surface allows for greater freedom in choosing substitutions. Additionally, the corresponding structural changes can be more predictable when altering residues on the protein surface compared to other positions, which is an advantage.

[0279] The substitution of one or more solvent-exposed residues is (E29K K30E E33K), N32G, N90T, K63R, T34V, T34K and T34R It can be selected from the group consisting of:

[0280] Please note that throughout the text, where groups of mutations are bracketed together (e.g., "(E29K K30E E33K)"), these mutations are, as appropriate, made together, i.e., simultaneously as a group / single option from a given list.

[0281] In particular, N32G results in improved helix stability.

[0282] In particular, K63R lengthens the chain, resulting in an increased hydrophobic area and rearrangement of charged atoms.

[0283] ·Partially buried type Mutating these residues advantageously adds additional hydrophobic mass / area to the core of the protein, which increases the amount of interactions in the protein core, which has the benefit of improving protein stability.

[0284] With regard to "aliphatic chain extension" mutations, they advantageously add larger side chains and also advantageously add additional hydrophobic mass to the core of the protein.

[0285] The substitution of one or more of the partially buried residues may be Q42E, T51I, T51V, T51L and T51F It can be selected from the group consisting of:

[0286] Additional advantageous properties shared by particular subgroups of substitutions are shown below: aliphatic chain extension T51I, T51L, T51F From amphiphilic to aliphatic T51I, T51V, T51L From amphiphilic to aromatic T51F From amines to acids Q42E

[0287] With respect to the Q42E mutation, this advantageously keeps the chain length of the amino acid residue the same and advantageously allows the change in chemical nature to be decoupled from changes to the overall size of the amino acid side chain.

[0288] Hydrophobic core (tertiary structure) These mutations have the advantage of increasing the burial of hydrophobic moieties and thus increasing stability.

[0289] One or more substitutions of hydrophobic core (tertiary structure) residues may be T45V, T45I, A59V, A59I, A59L, M65V and M65I It can be selected from the group consisting of:

[0290] Additional advantageous properties shared by particular subgroups of substitutions are shown below: chain extension T45I, A59V, A59I, A59L From amphiphilic to aliphatic T45V, T45I Beta branch M65V, M65I, A59V, A59I

[0291] Regarding "chain-extending" mutations, these add additional hydrophobic contacts and thus serve to further increase stability. This is particularly true for longer and / or branched side chains in the introduced amino acids. Furthermore, certain mutations remove polar groups from amino acid residues, resulting in entirely hydrophobic residues, whereas the wild type would be amphipathic.

[0292] Without wishing to be bound by theory, it is believed that the increased stability is due to the relative instability of exposed hydrophobic moieties in the unfolded state compared to buried hydrophobic side chains in the folded state. Thus, assuming good packing occurs in the core of the folded state, the more hydrophobic moieties there are, the more stable the native state is compared to the unfolded state. This is what we measure using Tm (a proxy for the Gibbs free energy of folding (DG)). Thus, the increased stability results from maximizing the amount of buried hydrophobicity through efficient packing of other hydrophobicity moieties.

[0293] Regarding both the partially buried and hydrophobic core (three-dimensional structure) mutation groups, they share the common property of being advantageous in that they tend to have a large number of contacts within the protein structure. The effect of targeting these residues is that a potentially larger effect on protein stability can be obtained with fewer substitutions compared to those targeting other parts of the protein. Thus, together, these mutation groups form a superset of substitutions that all share the same single special technical feature.

[0294] ·Secondary structure Note that the deletion of amino acid 61 converts the five-amino acid patch from residues 59-63 into a sequence consistent with a canonical β-turn motif. This four-amino acid motif, resulting from the deletion of amino acid 61, corresponds to a canonical β-turn motif. Our innovative approach utilizes existing residues in the wild-type protein to produce a substantial change in stability while allowing for minimal mutations to the starting polypeptide. The benefit provided by this group of mutations is that they stabilize a turn (and / or, in the case of N32G, a helix) in the protein. In either case, targeting these residues provides the advantage of promoting stable water binding throughout the affected region of the protein, thereby advantageously increasing stability.

[0295] The substitution of one or more secondary structure residues may be N32G, 59 AN-GK, 59 IN-GK, 59 VN-GK, 59 LN-GK, 59 AG-NK, 59 VG-NK, A59V, A59I, A59L, T34K and T34R It can be selected from the group consisting of:

[0296] Throughout the text, mutation groups are referred to as pairs (e.g., 59 When presented as "AN-GK"), these mutations are suitably made together, i.e., simultaneously as pairs / single choices from a given list.

[0297] Additional advantageous properties shared by particular subgroups of substitutions are shown below: C-terminal helix cap N32G, T34K, T34R turn A59V, A59I, A59L, 59 AN-GK, 59 IN-GK, 59 VN-GK, 59 LN-GK, 59AG-NK, 59 VG-NK Type I' (Type 1 Prime) 59 AN-GK, 59 IN-GK, 59 VN-GK, 59 LN-GK Type II' (Type 2 Prime) 59 AG-NK, 59 VG-NK Strands are on the rise T45V, T45I, M65V, M65I

[0298] ·Charge interaction These mutations offer the technical advantage of improving charge interactions and thereby increasing stability.

[0299] The substitution of one or more charge-interaction residues is Q42E, (E29K K30E E33K), T34K and T34R It can be selected from the group consisting of:

[0300] Additional advantageous properties shared by particular subgroups of substitutions are shown below: surface (E29K K30E E33K), T34K, T34R partially buried Q42E Acidic to basic and vice versa (E29K K30E E33K) From amines to acids Q42E From polar to basic T34R, T34K

[0301] In the particular case of Q42E, this improves the surface charge network. Without wishing to be bound by theory, it appears that Q42E adds charge to enable salt bridges (most likely to lysine residues) that would otherwise be too far away in the three-dimensional space of the protein to form such interactions. Salt bridges are typically considered to be charge-charge interactions between atoms that are 4 Å or less apart. Thus, this mutation appears to facilitate the formation of salt bridges from Q42E to Lys.

[0302] ·position Advantageously, the vast majority of substitutions taught herein are on the backside of the Stefin A-based polypeptide / scaffold protein, i.e., the non-binding end of the protein. This was surprising to the inventors. The expectation was that mutations would be scattered throughout the sequence of the polypeptide. However, the presented teachings allow for significant increases in stability by targeting the backside of the protein, leaving the binding end of the protein unmutated or minimally mutated.

[0303] One or more substitutions of residues at a particular position may be T51, A59, M65, N32, Q42, N90, K63, 59 AN-GK, 59 IN-GK, 59 VN-GK, 59 LN-GK, 59 AG-NK, 59 VG-NK and T34 It can be selected from the group consisting of:

[0304] If it is desired to limit mutations to particular positions within the polypeptide, this can be advantageously accomplished by selecting mutations from one of the individual groups set out below. Front face (joint end) T51. Back side (unbonded end) A59, M65, N32, Q42, N90, K63, 59 AN-GK,59 IN-GK, 59 VN-GK, 59 LN-GK, 59 AG-NK, 59 VG-NK and T34.

[0305] In this regard, it should be noted that in the global analysis performed by the inventors, every residue in the hydrophobic core of the protein was individually studied, and it was surprising to us that the most useful stability-enhancing mutations were concentrated in the back region of the protein.

[0306] Post-translational modifications An additional benefit of some of these mutations is that they promote the chemical stability of the molecule. For example, methionine residues can be oxidized, but valine / isoleucine residues cannot. Thus, the mutations taught by the inventors have the additional benefit of avoiding unwanted oxidation at this point in the protein, and therefore advantageously resulting in a molecule that is resistant to changes that might otherwise be caused by oxidation.

[0307] The substitution of one or more post-translationally modified residues may be N32G, M65V, M65I, T34V, T34K and T34R It can be selected from the group consisting of:

[0308] If it is desired to eliminate a particular type of post-translational modification, this can be advantageously accomplished by selecting a mutation from one of the individual groups shown below: Potential glycosylation N32G, T34V, T34K, T34R potential oxidation M65V, M65I

[0309] It is further noted that the N32G mutant also eliminates the risk of deamidation. Degradation of N residues by deamidation can be problematic - this mutation advantageously eliminates such a problem. The same applies to Q42E, as Q residues are also prone to deamidation. Potential deamidation N32G, Q42E

[0310] ·Difference between hSteA and SQT Note that N90T may be part of a turn in this region of the protein, and therefore provides the additional benefit of stabilizing that turn.

[0311] Exemplary Scaffolds Exemplary scaffolds for research applications: The scaffolding, named 3r (the r stands for research): 3r1 - hSteA Y35W N32G V48D M65I Q42E T51L (A59V ΔD61) (E29K K30E E33K) 3r2 - hSteA Y35W N32G V48D M65I Q42E T51L (A59V G60N ΔD61 N62G) (E29K K30E E33K)

[0312] Exemplary scaffolds for therapeutic applications: The scaffold, named 3t (the t stands for therapeutic): 3t1 - hSteA N32G V48D 3t2 - hSteA N32G V48D M65I 3t3 - hSteA N32G V48D M65I T51L 3t4 - hSteA N32G V48D M65I Q42E 3t5 - hSteA N32G V48D M65I Q42E T51L

[0313] In view of the standard / conventional nomenclature used herein, the complete scaffold protein sequences are disclosed with reference to SEQ ID NO: 1 and with respect to the stated mutations. However, for purposes of illustration, the following are representative of preferred scaffold protein sequences: 3r1 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 18 MIPGGLSEAKPATPEIQEIVDKVKPQLEKETGKTWGKLEAVEYKTQVD(Xn)GLNYYIKVRVGNKYIHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF 3r2 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 19 MIPGGLSEAKPATPEIQEIVDKVKPQLEKETGKTWGKLEAVEYKTQVD(Xn)GLNYYIKVRVNGKYIHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF 3t1 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 20 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD(Xn)GTNYYIKVRAGDNKYMHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF 3t2 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 21 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD(Xn)GTNYYIKVRAGDNKYIHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF 3t3 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 22 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD(Xn)GLNYYIKVRAGDNKYIHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF 3t4 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 23 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVEYKTQVD(Xn)GTNYYIKVRAGDNKYIHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF 3t5 with two heterologous peptide insertions. In this example, n=9: SEQ ID NO: 24 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVEYKTQVD(Xn)GLNYYIKVRAGDNKYIHLKVFKSL(Xn)EDLVLTGYQVDKNKDDELTGF In the sequence, X is any amino acid and n is an integer from 0 to 36. Most suitably, n is 9.

[0314] Cystatin A or Cystatin B Human stefin A belongs to family 1 of the cystatin superfamily. Cystatin A and cystatin B are distinct. Cystatin B typically has a near-neutral pI and contains a cysteine ​​residue near the C-terminus. It is believed that all cystatin Bs contain this cysteine ​​and all cystatin Bs form disulfide-linked dimers, whereas cystatin A has a more acidic pI and no cystatin A has this C-terminal cysteine.

[0315] Nucleic acids, promoters, libraries, and host cells The manufacture / production of recombinant polypeptides and / or nucleic acids according to the present invention is well known to those skilled in the art and requires only routine knowledge, such as how to synthesize polypeptides or polynucleotides and / or how to express polynucleotides to produce polypeptides in laboratory or large-scale commercial bioreactors. Numerous companies worldwide offer such routine production services; one need only provide them with the sequences to be produced.

[0316] Hosts, vectors for expression of polypeptides according to the invention, promoters for use in such systems, and codon optimization (if any) of the encoding nucleic acids are all well known to those skilled in the art. The choice of a particular vector, such as a phage, phagemid, or plasmid, or the choice of promoter or host cell or other such "tool" for the production of the polypeptides or libraries described herein is a matter for the skilled artisan working in the invention. Similarly, PCR or cloning strategies, ligation, transformation / electroporation techniques are all routine, do not form part of the present invention, and are determined by the operator.

[0317] If further guidance is needed, general molecular biology techniques are well known in the art, for example as in (2000 Current Protocols in Molecular Biology F.M. Ausubel et al, Eds. ISBN: 978-0-471-50338-5 published by John Wiley & Sons Ltd, Oldlands Way, Bognor Regis, West Sussex, PO22 9NQ, UK).

[0318] Exemplary cell strains: TG1 (Lucigen, Catalog No. 60502-2) ER2738 (New England Biolabs, Catalog No. E4104)

[0319] Exemplary phage strains: M13KO7 (New England Biolabs, Catalog No. N0315)

[0320] Exemplary phagemid vectors: pUC119 (Clontech, Catalog No. 3319), containing the lac promoter

[0321] Exemplary promoters: lac promoter (see above)

[0322] The polypeptides / scaffold proteins of the present invention may further comprise tags such as those for purification, for example a 6his tag, a maltose binding protein (MBP) tag, or any other suitable sequence to aid in purification.

[0323] The polypeptides / scaffold proteins of the invention can further comprise a linker, such as a glycine linker, for linking to another polypeptide.

[0324] The polypeptides / scaffold proteins of the invention may comprise a detection sequence, such as for detection by an antibody, for example a myc tag or a flag tag or any other suitable sequence to facilitate detection.

[0325] The polypeptides / scaffold proteins of the invention may be labeled, for example, a fluorescent label is attached to the polypeptide / scaffold protein.

[0326] The polypeptides / scaffold proteins of the invention can be linked to a carrier protein, such as a transport protein, to facilitate entry into cells.

[0327] The polypeptides / scaffold proteins of the invention can be linked to a targeting protein, such as an antibody or fragment thereof, or an aptamer, or an Affimer® reagent, to direct the polypeptides / scaffold proteins of the invention to a specific location, for example, for binding to a target cell or any other entity to which a targeted cell can bind.

[0328] The polypeptides / scaffold proteins of the invention can be attached to a substrate or structure, such as a bead, or nanosphere, or electrode (e.g., as part of an electrode array), or membrane (e.g., nitrocellulose membrane), or reaction vessel, such as an ELISA plate or microcentrifuge tube, or any other such article.

[0329] Suitably, the polypeptide / scaffold protein of the present invention may be immobilised.

[0330] Linkage of the polypeptide / scaffold protein to other moieties may be by any suitable means known in the art, for example by covalent linkage, by disulfide bridging, by preparation as a single polypeptide (fusion protein), by conjugation to a suitable amino acid residue such as a cysteine ​​residue, linkage to the N- or C-terminus of the polypeptide, or by any other suitable means known in the art.

[0331] Polypeptide production 1. A method of making a polypeptide that has altered stability, e.g., increased or decreased thermostability, compared to SEQ ID NO: 1, comprising: Compared to SEQ ID NO: 1, A12I, A12V, I16L, V20A, V20I, V20L, Q26E, E29M, T31K, N32G, N32D, N32H, T34V, T34R, T34K, T34D, T34P, L38A, L38V, L38F, A40I, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, G50S, T51F, T51V, T51L, T51I, T51A, A59L, A59I, A59V, K63R, M65V, M65I, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) and synthesizing a polypeptide comprising one or more substitutions selected from the group consisting of: More suitably, the substitution is A12I, A12V, I16L, V20I, V20L, Q26E, E29M, T31K N32G, N32D, N32H, T34V, T34R, T34D, T34P, L38A L38V, A40I, A40V, Q42D, T45I, T45V, V48E, V48G V48A, T51F, T51V, T51L, T51A, A59L, A59I, M65V L67I (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V) (E29K, K30E, E33K), (Y54D, T83D, Q86E) (A59L, G60N, D61G, N62K), (A59V, D61N, N62K) (G60N, D61G, N62K), (G60N, ΔD61, N62G) ΔD61, (A59L, G60N, ΔD61, N62G) (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G) (A59V, ΔD61), (G60P, ΔD61, N62P) (G60P, D61P, N62K), (G60P, ΔD61, N62G) (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E).

[0332] 1. A method of making a polypeptide with altered stability, e.g., increased thermal stability, compared to SEQ ID NO: 1, comprising: Compared to SEQ ID NO: 1, E29M, N32G, T34V, T34R, T34K, Q42E, T45I, T45V G50S, T51F, T51V, T51L, T51I, A59L, A59I, A59V K63R, M65V, M65I, L67I, N90T (E29K, K30E, E33K), (A59L, G60N, D61G, N62K) (A59V, D61N, N62K), (G60N, D61G, N62K) (G60N, ΔD61, N62G), ΔD61 (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K) (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G) synthesizing a polypeptide comprising one or more substitutions selected from the group consisting of (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61); More suitably, the substitution is E29M, N32G, T34V, T34R, T45I, T45V, T51F, T51V T51L, A59L, A59I, M65V, L67I (E29K, K30E, E33K), (A59L, G60N, D61G, N62K) (A59V, D61N, N62K), (G60N, D61G, N62K) (G60N, ΔD61, N62G), ΔD61 (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K) (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G) (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) Also disclosed are methods wherein the method is selected from the group consisting of:

[0333] 1. A method of making a polypeptide that has altered stability, e.g., decreased thermal stability, compared to SEQ ID NO: 1, comprising: Compared to SEQ ID NO: 1, A12I, A12V, I16L, V20A, V20I, V20L, Q26E, T31K N32D, N32H, T34D, T34P, L38A, L38V, L38F, A40I A40V, Q42D, V48E, V48D, V48G, V48A, V48L, T51A (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V) (Y54D, T83D, Q86E), (G60P, ΔD61, N62P) (G60P, D61P, N62K), (G60P, ΔD61, N62G) (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E), More suitably, the substitution is A12I, A12V, I16L, V20I, V20L, Q26E, T31K, N32D N32H, T34D, T34P, L38A, L38V, A40I, A40V, Q42D V48E, V48G, V48A, T51A (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V) (Y54D, T83D, Q86E), (G60P, ΔD61, N62P) (G60P, D61P, N62K), (G60P, ΔD61, N62G) (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E).

[0334] Suitably, the step of synthesizing comprises preparing nucleic acid encoding said polypeptide and orchestrating translation of said nucleic acid to produce the polypeptide. Suitably, the nucleic acid may be comprised in a phage genome, for example one or more members of a phage display library.

[0335] Additional mutations G4R, E18Q, P25S, N32Q, T34E, T34Q, G36E, M65F M65L, E78A, (K91E, D92K), (K91P, D93G), NPDG There may be additional benefits to making one or more additional mutations selected from the group consisting of:

[0336] Note that this group of mutations share the property that each contributes a small effect to thermostability. Care must be taken in measuring these, as when measured individually, experimental error in the measurements may yield a reading of a low effect, or even no effect or a small reversal of effect (e.g., if the experimental error in an individual measurement is within + / - 0.7°C of change in °C, it may indicate a zero or -0.2°C effect rather than a +0.5°C reading). This is true for all scientific measurements when experimental error is approximately equal to or exceeds the magnitude of the effect being measured.

[0337] With regard to the NPDG mutation beginning at amino acid 90 ("90NPDG"), this increases the statistical likelihood of forming a type 1 turn at this point in the protein, thus increasing stability by this mechanism.

[0338] Small effect - increased thermal stability G4R +0.05℃ E18Q +0.57℃ P25S +0.01℃ N32Q +0.37℃ T34Q +0.29℃ G36E +0.18℃ M65F +0.19℃ E78A +0.50℃ (K91E, D92K) +0.56℃ (K91P, D93G) +0.22℃ 90NPDG +0.7℃ (N90 K91P D92 D93G) +0.22℃

[0339] Small effect - reduced thermal stability T34E -0.39℃ M65L -0.23℃

[0340] PK effect In certain embodiments, a polypeptide, such as an Affimer polypeptide, comprises a polypeptide moiety, such as an Affimer polypeptide moiety, as a monomer that binds to a target moiety, preferably a protein, more preferably a human protein, and has a dissociation constant (K) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. D )

[0341] In certain embodiments, a polypeptide, such as an Affimer polypeptide, comprises a polypeptide moiety, such as an Affimer polypeptide moiety, as a monomer that binds to a target moiety, preferably a protein, more preferably a human protein, and has a length of about 10 -3 seconds -1 (i.e., in units of 1 / second) or slower, approximately 10 -4 seconds -1 or slower, or even about 10 -5 seconds -1or slower, e.g., as measured by Biacore, off )

[0342] In certain embodiments, a polypeptide, such as an Affimer polypeptide, comprises a polypeptide portion, such as an Affimer polypeptide portion, that binds to a target moiety, preferably a protein, more preferably a human protein, and has at least about 10 3 M -1 seconds -1 or faster, at least about 10 4 M -1 seconds -1 or faster, at least about 10 5 M -1 seconds -1 or faster, or even at least about 10 6 M -1 seconds -1 or faster, e.g., as measured by Biacore, on ) .

[0343] In certain embodiments, a polypeptide such as an Affimer polypeptide comprises a polypeptide moiety, such as an Affimer polypeptide moiety, as a monomer that binds to a target moiety, preferably a protein, more preferably a human protein, having a cognate binding partner, and has an IC50 of 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less in a competitive binding assay with the cognate binding partner.

[0344] advantage It is an advantage of the present invention that a scaffold is provided that has excellent expression characteristics.

[0345] It is an advantage of the present invention that a scaffold is provided that has little or reduced immunogenicity (low immunogenicity).

[0346] It is an advantage of the present invention that the described polypeptide / scaffold proteins do not suffer from aggregation / precipitation problems. More particularly, the scaffolds of the present invention are not observed to aggregate / precipitation before unfolding, and therefore, when a scaffold of the present invention has an increased Tm compared to hSteA, the scaffold also advantageously has increased resistance to aggregation / precipitation.

[0347] It is an advantage of the described polypeptide / scaffold proteins that they can accommodate heterologous peptide insertions.

[0348] It is an advantage of the polypeptide / scaffold proteins of the present invention that heterologous peptide inserts are properly presented.

[0349] It is an advantage of the present invention that the described polypeptides / scaffolds are not negatively affected in terms of stability / protease resistance by the mutations introduced. In other words, the polypeptides / scaffolds of the present invention advantageously retain their stability and / or protease resistance. This is another advantageous property of the particular mutations taught herein.

[0350] Further embodiments Also disclosed is the use of the above scaffolds in research applications, such as screening for the property of being able to bind to a particular target and / or screening for peptides with a particular activity(ies).

[0351] The use of the above scaffolds in medical applications, such as targeting compounds to specific cells or locations in the body, and / or in inhibiting or promoting specific metabolic activities, is also disclosed.

[0352] Also disclosed are compositions, such as pharmaceutical compositions, that comprise the above polypeptides / scaffold proteins.

[0353] Also disclosed are compositions comprising one or more of the polypeptides / scaffold proteins described above together with a pharmaceutically acceptable carrier, diluent or excipient.

[0354] Also disclosed is the use of the above polypeptide / scaffold proteins as diagnostic agents, therapeutic agents, biomarkers, agents that specifically detect biomarkers, rational drug design templates, targets or reagents for drug discovery, antibody surrogates, aptamers, Affimer® reagents, or research tools.

[0355] The use of the above polypeptides as scaffold proteins is also disclosed.

[0356] In one embodiment, a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1-11, 13-15, 17-19, 21-25, 27-28, 35-37, 39, 41, 43-44, 46-47, 49-50, 52-53, 55-58, 63-64, 66, 68-82, 84-85, and 87-98 of SEQ ID NO: 3 (canine wild-type SteA), Compared to SEQ ID NO: 3, T51L, T51V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q2 6E, E29M, T31K, N32D, N32H, T34V, T34R, T34D, T34P, A40V, Q42D, T45I, T45V, V48E , V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD 61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) A polypeptide is described which is characterized in that it contains one or more mutations selected from the group consisting of:

[0357] Suitably, said one or more mutations relative to SEQ ID NO: 1 are: T51L, T51V, M65V, N32G, A59I, E29M, T34V, T34R, T45I, T45V, T51F, A59L, L67I, (E29K , K30E, E33K), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G6 0N, ΔD61, N62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), and (A59V, ΔD61) and preferably said polypeptide has a Tm higher than the Tm of SEQ ID NO: 3, more preferably higher than the Tm of SEQ ID NO: 1. In another embodiment, suitably said one or more mutations relative to SEQ ID NO: 1 are selected from the group consisting of: L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q26E, T31K, N32D, N32H, T34D , T34P, A40V, Q42D, V48E, V48G, V48A, T51A, (V20I, L38A), (V20L, L38A), (V20 I, L38V), (V20L, L38V), (Y54D, T83D, Q86E), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E) and preferably said polypeptide has a Tm lower than the Tm of SEQ ID NO:3, more preferably lower than the Tm of SEQ ID NO:1.

[0358] In another embodiment, a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1-11, 13-15, 17-19, 21-25, 27-28, 35-37, 39, 41, 43-44, 46-47, 49-50, 52-53, 55-58, 63-64, 66, 68-82, 84-85, and 87-98 of SEQ ID NO: 3, and comprising at least one heterologous peptide insertion; The polypeptide, compared to SEQ ID NO: 1, M65I, T51I, T51L, T51V, M65V, A59V, N32G, A59I, L38A, V20A, V20I, A40I, L38V, G50S, L38F, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34K, T34D, T34P, A40V, Q42E, Q42D, T45I, T45V, V48E, V48D, V48G, V48A, V48L, T51F, T51A, A59L, K63R, L67I, N90T, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D , Q86E), (A59L, G60N, D61G, N62K), (A59V, D61N, N62K), (G60N, D61G, N62K), (G60N, ΔD61, N 62G), ΔD61, (A59L, G60N, ΔD61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K) , (A59I, G60N, ΔD61, N62G), (A59V, G60N, ΔD61, N62G), (A59V, ΔD61), (G60P, ΔD61, N62P), (G60P, D61P, N62K), (G60P, ΔD61, N62G), (G60P, D61G, N62K), (D61N, N62K), and (T83D, Q86E) and wherein the mutation comprises one or more mutations selected from the group consisting of: The heterologous peptide insertion is inserted at at least one of the following positions relative to SEQ ID NO: 1: d) 48-<heterologous peptide>-50, e) 49-<heterologous peptide>-51, f) 50-<heterologous peptide>-52, q) 72-<heterologous peptide>-77, r) 73-<heterologous peptide>-78, or s)74-<foreign peptide>-79 A polypeptide is described, comprising: [Brief explanation of the drawings]

[0359] [Figure 1] FIG. [Figure 2] FIG. [Figure 3] FIG. [Figure 4A-4B] FIG. [Figure 5] FIG. [Figure 6] FIG. [Figure 7] FIG. [Figure 8] FIG. [Figure 9] FIG. [Figure 10] FIG. [Figure 11] FIG. [Figure 12] FIG. [Figure 13] FIG. [Figure 14] FIG. [Figure 15] FIG. [Figure 16] FIG. [Figure 17] FIG. [Figure 18] FIG. [Figure 19] FIG. DETAILED DESCRIPTION OF THE INVENTION

[0360] I. Definition To facilitate the understanding of the present invention, several terms and phrases are defined below.

[0361] The term "Affimer" (or "Affimer polypeptide") refers to small, highly stable proteins that are recombinantly engineered variants of stefin polypeptides. Affimer proteins exhibit peptide loops (usually two) and N-terminal sequences, all of which can be randomly selected to bind desired target proteins with high affinity and specificity, similar to monoclonal antibodies. Stabilization of the peptide loops by the stefin protein scaffold constrains the possible conformations that the peptides can adopt, thus increasing binding affinity and specificity compared to libraries of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition properties of monoclonal antibodies in various applications. Changes can be made in other parts of the stefin polypeptide sequence to improve the properties of these affinity reagents, for example, increasing their stability, making them robust over a variety of temperature and pH ranges, and the like.

[0362] "Encoded Affimer" refers to a nucleic acid construct that, when expressed by cells within a patient's body by a gene delivery process, produces the Affimer polypeptide of interest in vivo.

[0363] An "Affimer-linked conjugate" refers to an Affimer polypeptide to which one or more moieties have been conjugated by chemical conjugation, rather than by forming a contiguous peptide linked through the C-terminus or N-terminus of the polypeptide portion of the Affimer polypeptide containing the Affimer polypeptide sequence. An Affimer-linked conjugate may also be an "Affimer-drug conjugate," which refers to an Affimer polypeptide containing one or more pharmacologically active moieties conjugated thereto. An Affimer-linked conjugate may also be an "Affimer-tag conjugate," which refers to an Affimer polypeptide containing one or more detectable moieties (i.e., detectable labels) conjugated thereto.

[0364] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified, naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as polypeptides containing other modifications known in the art.

[0365] The terms "amino acid residue" and "amino acid" are used interchangeably and, in the context of a polypeptide, refer to an amino acid that participates in one or more peptide bonds of the polypeptide. In general, the abbreviations used herein to designate amino acids are based on the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission (see Biochemistry (1972) 11:1726-1732). For example, Met, Ile, Leu, Ala, and Gly represent "residues" of methionine, isoleucine, leucine, alanine, and glycine, respectively. Residue refers to the radical derived from the corresponding α-amino acid by removing the OH moiety of the carboxyl group and the H moiety of the α-amino group. The term "amino acid side chain" refers to the portion of an amino acid that does not contain the --CH(NH2)COOH moiety, as defined by KD Kopple, "Peptides and Amino Acids," WA Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.

[0366] In most cases, the amino acids used in the applications of the present invention will be naturally occurring amino acids found in proteins or naturally occurring anabolic or catabolic products of such amino acids containing amino and carboxyl groups. Particularly suitable amino acid side chains include those selected from the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, as well as amino acids and amino acid analogs identified as components of peptidoglycan bacterial cell walls.

[0367] Amino acid residues with "basic side chains" include Arg, Lys, and His. Amino acid residues with "acidic side chains" include Glu and Asp. Amino acid residues with "neutral polar side chains" include Ser, Thr, Asn, Gln, Cys, and Tyr. Amino acid residues with "neutral nonpolar side chains" include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp, and Phe. Amino acid residues with "nonpolar aliphatic side chains" include Gly, Ala, Val, Ile, and Leu. Amino acid residues with "hydrophobic side chains" include Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp. Amino acid residues with "small hydrophobic side chains" include Ala and Val. Amino acid residues with "aromatic side chains" include Tyr, Trp, and Phe.

[0368] The term amino acid residue further includes analogs, derivatives, and homologs of any specific amino acid referred to herein. For example, a subject polypeptide such as Affimer (especially when produced by chemical synthesis) may contain amino acid analogs such as cyanoalanine, canavanine, dienecholic acid, norleucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having suitable side chains as used herein will be apparent to those skilled in the art and are within the scope of the present invention.

[0369] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), not counting any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning that they share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, e.g., at least 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies. In some embodiments, the identity exists over a region of nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60-80 bases, e.g., over at least 80-100 bases or 101 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., over the nucleotide sequence encoding the desired protein.

[0370] A "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are generally defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conservative substitution. In general, conservative substitutions in the sequence of a polypeptide, soluble protein, and / or antibody of the invention do not abolish binding of the polypeptide, soluble protein, or antibody containing that amino acid sequence to its target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0371] An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0372] The term "substantially pure," as used herein, refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0373] The term "fusion protein" or "fusion polypeptide," as used herein, refers to a hybrid protein expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

[0374] The term "linker" or "linker region," as used herein, refers to a linker inserted between a first polypeptide (e.g., a copy of an Affimer) and a second polypeptide (e.g., another Affimer, an Fc domain, a ligand-binding domain, etc.). In some embodiments, the linker is a peptide linker. The linker should not adversely affect the expression, secretion, or bioactivity of the polypeptide. Preferably, the linker is not antigenic and does not elicit an immune response.

[0375] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0376] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of nucleotides along a strand of deoxyribonucleic acid deoxyribonucleotides. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain and thus the nucleic acid sequence that encodes the amino acid sequence.

[0377] As used herein, "sequence," when used in reference to a nucleotide sequence, grammatical and other forms of this term, can include DNA or RNA, and can be single-stranded or double-stranded. Nucleic acid sequences can also be mutated. Nucleic acid sequences can be of any length, e.g., from 2 to 100,000 or 100,001 or more nucleotides (or any integer value above or therebetween), and nucleic acids can have lengths of, e.g., from about 100 to about 10,000, or from about 200 to about 500 nucleotides.

[0378] The term "vector," as used herein, refers to a construct that can deliver one or more desired genes or sequences into a host cell and typically express them in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0379] As used herein, the term "transfection" refers to the transfer of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistic technology (particle bombardment).

[0380] The term "carrier," as used herein, refers to an isolated nucleic acid, including an isolated nucleic acid that can be used to deliver a composition to the interior of a cell. Several carriers are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds, such as polylysine compounds, liposomes, and the like, that facilitate the transfer of nucleic acids into cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0381] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an operably linked expression control sequence and a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements used for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0382] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence and a second nucleic acid sequence are operably linked when they are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, if necessary, join two protein-coding regions in the same reading frame.

[0383] As used herein, the term "promoter" is defined as a promoter DNA sequence recognized by the synthetic machinery required for or directed to the synthetic machinery for cell-specific transcription of a polynucleotide sequence.

[0384] The term "constitutive expression" as used herein refers to all that is expressed under physiological conditions.

[0385] The term "inducible expression," as used herein, refers to expression under certain conditions, such as activation (or inactivation) of an intracellular signaling pathway or contact of a cell harboring an expression construct with a small molecule that modulates expression (or expression level) of a gene operably linked to an inducible promoter that is sensitive to the concentration of the small molecule.

[0386] The term "electroporation" refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows biomolecules, such as plasmids or other oligonucleotides, to pass from one side of the cell membrane to the other.

[0387] Suitably, a polypeptide of the present invention comprises an amino acid sequence as described herein. Suitably, a polypeptide of the present invention consists essentially of an amino acid sequence as described herein. Suitably, a polypeptide of the present invention consists of an amino acid sequence as described herein.

[0388] II. Fusion Proteins - Overview In some embodiments, an Affimer polypeptide can further comprise one or more additional polypeptide sequences at one or both ends of the Affimer sequence that modulate the biological activity of the Affimer polypeptide. For example, these additions can modulate one or more properties of the modified Affimer, such as affinity, e.g., for binding to and inhibiting a target molecule, modulate circulating half-life, modulate therapeutic drug half-life, modulate stability of the Affimer polypeptide, modulate protease cleavage, modulate dosage, modulate release or bioavailability, facilitate purification, reduce deamidation, improve shelf life, or improve or alter a particular route of administration. Similarly, Affimer polypeptides can include a protease cleavage sequence, a reactive group, an antibody binding domain (including but not limited to FLAG or polyHis), or other affinity-based sequences (including but not limited to FLAG, polyHis, GST, etc.), or an attached molecule (including but not limited to biotin) that improves detection, purification, or other characteristics of the polypeptide.

[0389] Thus, in certain embodiments of the invention, the Affimer polypeptide is a fusion protein (herein "fusion domain") having at least one Affimer polypeptide sequence and one or more heterologous polypeptide sequences. By way of example only, the fusion domain can be selected to confer desired properties, such as secretion from cells or cell surface retention (i.e., for the encoded Affimer), to serve as a substrate or other recognition sequence for post-translational modification, to create multimeric structures that aggregate through protein-protein interactions, to alter (often extend) serum half-life, or to alter tissue localization or clearance and other ADME properties.

[0390] For example, some fusion domains are particularly useful for isolation and / or purification of the fusion protein, such as by affinity chromatography. Well-known examples of such fusion domains that facilitate expression or purification include, by way of illustration only, affinity tags, such as polyhistidine (i.e., His6 tag), Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S tag, HA tag, c-Myc tag, thioredoxin, protein A, and protein G.

[0391] Affiner polypeptides generally contain a signal sequence that directs transport of the protein into the lumen of the endoplasmic reticulum for secretion, ultimately resulting in secretion (or retention at the cell surface in the case of a transmembrane domain or other cell surface retention signal). Signal sequences (also called signal peptides or leader sequences) are located at the N-terminus of nascent polypeptides. They target polypeptides to the endoplasmic reticulum, where proteins are sorted to their destination, for example, the internal space of an organelle, an intracellular membrane, the outer membrane of the cell, or the outside of the cell via secretion. Most signal sequences are cleaved from proteins by signal peptidases after the protein is transported to the endoplasmic reticulum. Cleavage of a signal sequence from a polypeptide usually occurs at a specific site in the amino acid sequence and depends on the amino acid residues in the signal sequence.

[0392] In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (e.g., about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).

[0393] In some embodiments, the signal peptide is a native signal peptide from a human protein. In other embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a mutant native signal peptide from the corresponding natively secreted human protein, which may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions, insertions, or deletions.

[0394] In some embodiments, the signal peptide is a signal peptide or mutant thereof from an immunoglobulin (e.g., IgG heavy chain or IgG-kappa light chain), a cytokine (e.g., interleukin-2 (IL-2) or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide that enables efficient secretion of a protein from a cell. Exemplary signal peptides include, but are not limited to, the following:

[0395] [Table 10]

[0396] In some embodiments of a secreted Affimer polypeptide, the recombinant polypeptide includes a signal peptide when expressed, and this signal peptide (or a portion thereof) is cleaved from the Affimer polypeptide upon secretion.

[0397] The subject fusion proteins can also include one or more linkers separating heterologous protein sequences or domains. As used herein, the term "linker" refers to a linker amino acid sequence inserted between a first polypeptide (e.g., an affimer) and a second polypeptide (e.g., a second affimer, an Fc region, a receptor trap, albumin, etc.). Experimental linkers designed by researchers are generally classified into three categories according to their structure: flexible linkers, rigid linkers, and in vivo cleavable linkers. In addition to the essential role of linking functional domains together (as in flexible and rigid linkers) or releasing free functional domains in vivo (as in in vivo cleavable linkers), linkers offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving a desirable pharmacokinetic profile. Linkers should not adversely affect the expression, secretion, or biological activity of the fusion protein. The linker should not be antigenic and should not provoke an immune response.

[0398] Suitable linkers are known to those of skill in the art and often contain a mixture of glycine and serine residues, often including sterically unhindered amino acids. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example, from 1 to 50 amino acids, 1 to 22 amino acids, 1 to 10 amino acids, 1 to 5 amino acids, or 1 to 3 amino acids. In some embodiments, the linker can include a cleavage site. In some embodiments, the linker can include an enzymatic cleavage site, thereby separating the second polypeptide from the first polypeptide.

[0399] In certain preferred embodiments, the linker can be characterized as flexible. Flexible linkers are typically applied when some degree of movement or interaction is desired between the linked domains. They are generally composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. See, e.g., Argos P. (1990) "An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion" J Mol Biol. 211:943-958. The small size of these amino acids provides flexibility, allowing mobility of the connected functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the protein moiety. The most commonly used flexible linkers have a sequence consisting primarily of a stretch of Gly and Ser residues ("GS" linker). One example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser). By adjusting the copy number "n," the length of this GS linker can be optimized to achieve appropriate separation of functional domains or maintain necessary interdomain interactions, and the length of this GS linker is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers also contain many small or polar amino acids such as Gly and Ser, but can also contain additional amino acids such as Thr and Ala to maintain flexibility and polar amino acids such as Lys and Glu to improve solubility.

[0400] In certain preferred embodiments, the linker can be characterized as rigid. Flexible linkers have the advantage of passively connecting functional domains and allowing some mobility, but the lack of rigidity of these linkers can limit certain fusion protein embodiments, for example, in expression yield or biological activity. The ineffectiveness of flexible linkers in these cases results from insufficient isolation of the protein domains or insufficient reduction of their interference with each other. In these situations, rigid linkers have been successfully applied to maintain a fixed distance between the domains and to maintain their independent functions.

[0401] Many natural linkers exhibit α-helical structures. α-helical structures are rigid and stable due to intersegment hydrogen bonds and tightly packed backbones. Therefore, rigid α-helical linkers can act as rigid spacers between protein domains. George et al. (2002) "An analysis of protein domain linkers: their classification and role in protein folding" Protein Eng. 15(11):871-9. Generally, rigid linkers exhibit a relatively rigid structure by adopting an α-helical structure or by containing multiple Pro residues. Under many circumstances, they separate functional domains more efficiently than flexible linkers. The length of the linker can be easily adjusted by varying the copy number to achieve the optimal distance between domains. Consequently, rigid linkers are selected when spatial separation of domains is essential to preserve the stability or biological activity of the fusion protein. In this regard, alpha-helix-forming linkers having the sequence (EAAAK)n, where n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, have been applied in the construction of many recombinant fusion proteins. Another type of rigid linker has a Pro-rich sequence (XP)n, where X represents any amino acid, preferably Ala, Lys, or Glu, and n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0402] For illustrative purposes only, exemplary linkers include:

[0403] [Table 11]

[0404] Other linkers that can be used in the subject fusion proteins include, but are not limited to, SerGly, GGSG (SEQ ID NO:59), GSGS (SEQ ID NO:60), GGGS (SEQ ID NO:61), S(GGS)n (SEQ ID NO:62) (n is 1 to 7), GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO:63), ESGGGGVT (SEQ ID NO:64), LESGGGGVT (SEQ ID NO:65), GRAQVT (SEQ ID NO:66), WRAQVT (SEQ ID NO:67), and ARGRAQVT (SEQ ID NO:68). The hinge region of the Fc fusions described below can also be considered a linker.

[0405] Various elements can be used to anchor proteins to the plasma membrane of cells. For example, the transmembrane (TM) domains of type I (N-terminus facing outside the cell) and type II (N-terminus facing into the cytosol) integral membrane proteins can be used to target chimeric proteins to the plasma membrane. Proteins can also be attached to the cell surface by fusing a glycosylphosphatidylinositol (GPI) lipid signal to the 3' end of the gene. Cleavage of a short carboxy-terminal peptide allows for the attachment of glycolipids to the newly exposed C-terminus via an amide linkage. See Udenfriend et al. (1995) "How Glycosylphoshpatidylinositol Anchored Membrane Proteins are Made" Annu Rev Biochem 64:563-591.

[0406] In certain embodiments, the fusion protein contains a transmembrane polypeptide sequence (transmembrane domain). A distinguishing feature of a suitable transmembrane polypeptide is its ability to be expressed on the surface of a cell, allowing the Affimer polypeptide to be displayed on the surface of the cell. In certain embodiments, this may be an immune cell, particularly a lymphocyte or natural killer (NK) cell, where upon expression of the cell surface feature upon interaction with a tumor cell, the Affimer polypeptide on the immune cell surface binds thereto and directs the immune cell's cellular response against the designated target tumor cell. The transmembrane domain may be derived from natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. By way of non-limiting example, the transmembrane polypeptide may be a subunit of a T cell receptor, such as α, β, γ, or δ; a polypeptide constituting the CD3 complex; the IL2 receptor p55 (α chain), p75 (β chain), or γ chain; a subunit chain of an Fc receptor, particularly Fey receptor III, or a CD protein. Alternatively, the transmembrane domain may be synthetic and comprise predominantly hydrophobic residues such as leucine and valine.

[0407] In certain other embodiments, the Affimer polypeptide is a fusion protein that includes, in addition to the Affimer polypeptide, a sequence that transmits a signal for the post-translational addition of a glycosylphosphatidylinositol (GPI) anchor. A GPI anchor is a glycolipid structure that is post-translationally added to the C-terminus of many eukaryotic proteins. This modification to the Affimer polypeptide tethers (attaches) it to the extracellular surface of the plasma membrane of a cell in which the Affimer polypeptide is re-expressed as a recombinant protein (i.e., the encoded Affimer described below). In these embodiments, the GPI anchor domain is C-terminal to the Affimer polypeptide sequence, preferably at the C-terminus of the fusion protein.

[0408] In one embodiment, the GPI anchor domain is a polypeptide that transmits a signal for post-translational addition of a GPI anchor when the fusion protein of which it is a part is expressed in a eukaryotic cell system. The GPI anchor signal sequence consists of a series of small amino acids at the anchor attachment site (ω site) followed by a hydrophobic spacer and ending in a hydrophobic stretch (Low, (1989) FASEB J. 3:1600-1608). Cleavage of this signal sequence occurs in the ER before anchor attachment, and while the central components are conserved, the peripheral portion is variable (Homans et al., Nature, 333:269-272 (1988)). The C-terminus of GPI-tethered proteins is linked to the highly conserved core glycan mannose(α1-2)mannose(α1-6)mannose(α1-4)glucosamine(α1-6)myoinositol by a phosphoethanolamine bridge. A phospholipid tail connects the GPI anchor to the cell membrane.

[0409] Exemplary GPI anchor domains that can be used in the subject Affimer-containing fusion proteins include the following: SEQ ID NO: 69 SGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT SEQ ID NO: 70 SGTSPGLSAGATVGIMIGVLVGVALI SEQ ID NO: 71 SAPVLSAVATVGITIGVLARVALI SEQ ID NO: 72 SSPDLSAGTAVSIMIGVLAGMALI SEQ ID NO: 73 TLGGNSASYTFVSLLFSAVTLLLLC SEQ ID NO: 74 SGTSPGLSAGATVGIMIGVLVGVALI

[0410] GPI anchor attachment can be achieved by expression of Affimer fusion proteins containing a GPI anchor domain in eukaryotic cell lines capable of GPI post-translational modification. As with transmembrane domain fusion proteins, human cells, including lymphocytes or other cells involved in tumor initiation or promotion, are competent to express and can be engineered to express an encoded Affimer containing a GPI anchor domain in order to retain the expressed Affimer-containing fusion on the surface of the engineered cells.

[0411] Yet another modification that can be made to the Affimer polypeptide sequence itself or to an adjacent polypeptide portion provided as part of a fusion protein is one or more sequences that are sites for enzymatic post-translational modifications, including, but not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linked modifications, and the like.

[0412] a. Modification of PK and ADME properties In certain embodiments, Affimer polypeptides may not have a half-life and / or PK profile that is optimal for routes of administration, such as parenteral therapeutic administration. The term "half-life" refers to the amount of time it takes for a substance, such as an Affimer polypeptide of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the substance's excretion, excretion, degradation (e.g., enzymatic), or absorption and concentration in a particular organ or tissue of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the substance's plasma concentration to reach half of its steady-state level ("serum half-life"). To address this shortcoming, there are various general strategies for extending half-life that have been used in the case of other protein therapeutics, including incorporating a half-life extending moiety as part of the Affimer polypeptide.

[0413] The term "half-life extending moiety" refers to a pharmaceutically acceptable moiety, domain, or molecule that is covalently linked ("conjugated" or "fused") to an Affimer polypeptide, either via a non-naturally encoded amino acid, directly, or via a linker, to form an Affimer polypeptide described herein, and that prevents or attenuates modifications that reduce in vivo proteolysis or other activity of the Affimer polypeptide, increases half-life, and / or improves or alters other pharmacological or biophysical properties, including, but not limited to, increasing the absorption rate of the modified Affimer polypeptide, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and / or target selectivity, increasing manufacturability and / or reducing immunogenicity of the modified Affimer polypeptide, compared to a control such as the unconjugated form of the modified Affimer polypeptide. The term "half-life extending moiety" includes non-proteinaceous half-life extending moieties, such as water soluble polymers, e.g., polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and branched or unbranched C8-C30 alkyl groups; and proteinaceous half-life extending moieties, such as serum albumin, transferrin, Adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) Adnectins), Fc domains, and unstructured proteins, such as XTEN and PAS polypeptides (e.g., conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and / or Ser), and fragments of any of the foregoing.

[0414] In certain aspects, the half-life extending moiety extends the circulating half-life of the resulting Affimer polypeptide in mammalian serum compared to the half-life of the protein not so conjugated to the moiety (e.g., compared to the Affimer polypeptide alone). In some embodiments, the half-life is extended by more than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety.

[0415] By way of further example, half-life extending moieties that can be used in producing the Affimer polypeptides of the present invention include: Genetic fusion of a pharmacologically active Affimer sequence with a naturally long half-life protein or protein domain (e.g., Fc fusion, transferrin [Tf:transferrin] fusion, or albumin fusion). For example, Beck et al. (2011) “Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs. 3:1-2; Czajkowsky et al. (2012) “Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 4:1015-28; Huang et al. Mimetibody technology” Curr Opin Biotechnol. 2009;20:692-9; Keefe et al. (2013) “Transferrin fusion protein therapies: acetylcholine receptor-transferrin fusion protein as a model. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; p. 345-56; Weimer et al. (2013) “Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges.Hoboken: Wiley; 2013. p. 297-323; Walker et al.(2013) "Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. See pp. 325-43.

[0416] Genetic fusion of a pharmacologically active Affimer sequence with an inert polypeptide, such as XTEN (also known as recombinant PEG or "rPEG"), homoamino acid polymer (HAP; HAP-ylated), proline-alanine-serine polymer (PAS; PAS-ylated), or elastin-like peptide (ELP; ELP-ylated).For example, see Schellenberger et al. (2009) “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009;27:1186-90; Schlapschy et al. Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007;20:273-84; Schlapschy (2013) PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel. 26:489-501. Floss et al. (2012) “Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnol. 28:37-45.Floss et al. “ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. p. 372-98.

[0417] Chemical conjugation of pharmacologically active peptides or proteins with repetitive chemical moieties, for example PEG (PEGylation) or hyaluronic acid, to increase the hydrodynamic radius. For example, Caliceti et al. (2003) “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates” Adv Drug Delivery Rev. 55:1261-77; Jevsevar et al. (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113-28; Kontermann (2009) “Strategies to extend plasma half-lives of See Kang et al. (2009) “Emerging PEGylated drugs” Expert Opin Emerg Drugs. 14:363-80; and Mero et al. (2013) “Conjugation of hyaluronan to proteins” Carb Polymers. 92:2163-70.

[0418] (b) Significantly increasing the negative charge of the fusion of a pharmacologically active peptide or protein by polysialylation, or alternatively, fusing a negatively charged, highly sialylated peptide (e.g., carboxy-terminal peptide [CTP; of the chorionic gonadotropin (CG) b chain]) with a biologic candidate, which is known to extend the half-life of native proteins such as the human CG b-subunit. For example, Gregoriadis et al. (2005) “Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids” Int J Pharm. 2005; 300:125-30; Duijkers et al. “Single dose pharmacokinetics and effects on follicular growth and hormonal serums of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary-suppressed females” (2002) Hum Reprod. 17:1987-93; and Fares et al. extension through See O-glycosylation.

[0419] Conjugation of peptide or protein binding domains to biologically active proteins, resulting in non-covalent binding to proteins that typically have a long half-life, such as HSA, human IgG, transferrin or fibronectin. For example, Andersen et al. (2011) “Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain” J Biol Chem. 286:5234-41; O'Connor-Semmes et al. (2014) “GSK2374697, a novel albumin-binding domain antibody (albudAb), extends systemic exposure of extendin-4: first study in See “humans-PK / PD and safety” Clin Pharmacol Ther. 2014;96:704-12. Sockolosky et al. (2014) “Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice” PLoS One. 2014;9:e102566.

[0420] Classical genetic fusion with long-lived serum proteins offers an alternative half-life extension method that is distinct from chemical conjugation with PEG or lipids. Two major proteins have traditionally been used as fusion partners: the antibody Fc domain and human serum albumin (HSA). Fc fusion involves fusing a peptide, protein, or receptor extracellular domain to the Fc portion of an antibody. Both Fc and albumin fusions achieve half-life extension not only by increasing the size of peptide drugs, but also by utilizing the body's natural recycling mechanism: the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in endosomes. Fusions based on these proteins can have half-lives ranging from 3 to 16 days, much longer than typical PEGylated or lipidated peptides. Fusion with an antibody Fc domain can improve the solubility and stability of peptide or protein drugs. One example of a peptide-Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, a protein utilized by fatty acylated peptides, is another common fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. A major difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA, resulting in the presentation of the fusion peptide as a dimer or monomer, depending on the choice of fusion partner. The dimeric nature of Affimer-Fc fusions can produce avidity effects when Affimer targets, such as cell surface proteins on target cells, are sufficiently close together or are themselves dimers or higher-order multimers. This may be desirable or target-independent.

[0421] (i)Fc fusion In some embodiments, an Affimer polypeptide can be a fusion protein with an immunoglobulin Fc domain ("Fc domain"), or a portion of a fragment or variant thereof, e.g., a functional Fc region. In this regard, an Affimer polypeptide created as an Fc fusion ("Fc-fusion"), e.g., an Affimer-Fc fusion protein, is a polypeptide comprising one or more Affimer sequences covalently linked (directly or indirectly) to an immunoglobulin Fc region by a peptide backbone. An Fc-fusion can, for example, comprise an antibody Fc region (which facilitates effector function and pharmacokinetics) and an Affimer sequence(s) as part of the same polypeptide. The immunoglobulin Fc region can also be indirectly linked to one or more Affimers. A variety of linkers are known in the art and may be used to link Fc to a polypeptide comprising an Affimer sequence to generate an Fc-fusion. In certain embodiments, Fc-fusions may be dimerized to form Fc-fusion homodimers or, using non-identical Fc domains, to form Fc-fusion heterodimers.

[0422] There are several reasons for selecting the Fc region of a human antibody for use in producing the subject Affimer polypeptide as an Affimer fusion protein. The primary rationale is to produce a stable protein large enough to exhibit a similar pharmacokinetic profile to that of an antibody and to take advantage of the properties conferred by the Fc region; this includes the salvage neonatal FcRn receptor pathway, which involves FcRn-mediated recycling of the fusion protein to the cell surface after endocytosis, resulting in its escape from lysosomal degradation and release back into the bloodstream, thus contributing to its extended serum half-life. Another obvious advantage is the attachment of the Fc domain to Protein A, which can simplify downstream processing during production of the Affimer polypeptide and enable the generation of highly pure preparations of the Affimer polypeptide.

[0423] Generally, the Fc domain will include the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, the Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, plus the flexible hinge N-terminal to these domains. For IgA and IgM, the Fc may include the J chain. For IgG, the Fc includes immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domains can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 carboxyl-terminal to it, with numbering according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region alone or in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at several different Fc positions, and these are also included as Fc domains as used herein.

[0424] In certain embodiments, Fc, as used herein, "functional Fc region" refers to an Fc domain or fragment thereof that retains the ability to bind to FcRn. A functional Fc region binds to FcRn but does not have effector function. The ability of an Fc region or a fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions.

[0425] In an exemplary embodiment, the Fc domain is derived from the IgG1 subclass, although other subclasses (e.g., IgG2, IgG3, and IgG4) can also be used. An exemplary sequence of a human IgG1 immunoglobulin Fc domain that can be used is: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 75) is.

[0426] In some embodiments, the Fc region used in the fusion protein can comprise the hinge region of an Fc molecule. An exemplary hinge region comprises core hinge residues spanning positions 1-16 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above (i.e., DKTHTCPPCPAPELLG (SEQ ID NO: 76)). In certain embodiments, the Affimer-containing fusion protein can adopt a multimeric structure (e.g., a dimer) due, in part, to cysteine ​​residues at positions 6 and 9 within the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In other embodiments, the hinge region as used herein can further comprise residues from the CH1 and CH2 regions adjacent to the core hinge sequence of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In yet other embodiments, the hinge sequence can comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 77) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 78).

[0427] In some embodiments, the hinge sequence can include one or more substitutions that confer desirable pharmacokinetic, biophysical and / or biological properties. Some exemplary hinge sequences include: SEQ ID NO: 79 EPKSCDKTHTCPPCPAPELLGGPS SEQ ID NO:80 EPKSSDKTHTCPPCPAPELLGGPS; SEQ ID NO:81 EPKSSDKTHTCPPCPAPELLGGSS; SEQ ID NO:82 EPKSSGSTHTCPPCPAPELLGGSS; SEQ ID NO: 83 DKTHTCPPCPAPELLGGPS and SEQ ID NO: 84 DKTHTCPPCPAPELLGGSS Examples include:

[0428] In one embodiment, residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above can be replaced with S to eliminate Fc effector function, as exemplified in hinges having the sequences EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 85), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 86), and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 87). In another embodiment, residue DK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above can be replaced with GS to remove a potential clip site, as exemplified in the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 88). In another embodiment, the C at position 103 of the heavy chain constant region of human IgG1 (i.e., domains CH1-CH3) can be replaced with an S to prevent inappropriate cysteine ​​bond formation in the absence of the light chain; this replacement is exemplified by EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 89), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 90), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 91).

[0429] In some embodiments, the Fc is a mammalian Fc, such as a human Fc, comprising an Fc domain derived from IgG1, IgG2, IgG3, or IgG4. The Fc region can have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a native Fc region and / or with the Fc region of the parent polypeptide. In some embodiments, the Fc region can have at least about 90% sequence identity with a native Fc region and / or with the Fc region of the parent polypeptide.

[0430] In some embodiments, the Fc domain comprises an amino acid sequence selected from the examples provided by SEQ ID NOs: 4-16. It should be understood that the C-terminal lysine of the Fc domain is an optional component of a fusion protein comprising an Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 4-16, excluding the C-terminal lysine.

[0431] [Table 12] TIFF0007659292000020.tif221165

[0432] The Affimer sequence can be placed at either the N-terminus or C-terminus of the Fc domain and may be directly linked, or the fusion protein may have another polypeptide sequence located between the Fc domain and the Affimer polypeptide sequence.

[0433] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binding to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins.

[0434] In certain embodiments, the fusion protein comprises an Fc domain sequence, such that the resulting Affimer polypeptide lacks (or has reduced) ADCC and / or complement activation or effector functionality. For example, the Fc domain may comprise a naturally inactive constant region of an IgG2 or IgG4 isotype, or a mutated IgG1 constant region. Examples of suitable modifications are described in EP 0307434. One example includes the substitution of alanine residues at positions 235 and 237 (EU index numbering).

[0435] In other embodiments, the fusion protein comprises an Fc domain sequence, so that the resulting Affimer polypeptide will retain some or all of its functionality, e.g., it will be capable of ADCC and / or CDC activity, such as when the fusion protein comprises an Fc domain from human IgG1 or IgG3. The level of effector function can be altered according to known techniques, for example by mutations in the CH2 domain (e.g., when an IgG1 CH2 domain has one or more mutations at positions selected from 239, 332, and 330, e.g., the mutations are selected from S239D, I332E, and A330L, so that the antibody has enhanced effector function), and / or by modifying the glycosylation profile of the antigen-binding protein of the invention so that there is reduced fucosylation in the Fc region.

[0436] (ii) Albumin fusions In other embodiments, the Affimer polypeptide is a fusion protein comprising at least one Affimer sequence plus an albumin sequence or albumin fragment. In other embodiments, the Affimer polypeptide is conjugated to the albumin sequence or albumin fragment by chemical linkage other than incorporation into the Affimer-containing polypeptide sequence. In some embodiments, the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment. Albumin serum proteins equivalent to HSA are found, for example, in cynomolgus monkeys, cows, dogs, rabbits, and rats. Among non-human species, bovine serum albumin (BSA) is most structurally similar to HSA. See, e.g., Kosa et al., (2007) J Pharm Sci. 96(11):3117-24. The present disclosure contemplates the use of albumins from non-human species, including, but not limited to, albumin sequences derived from cynomolgus monkey serum albumin or bovine serum albumin.

[0437] Mature HSA, a 585-amino acid polypeptide (approximately 67 kDa) with a serum half-life of approximately 20 days, is primarily responsible for blood colloid oncotic pressure, blood pH, and the transport and distribution of numerous endogenous and exogenous ligands. The protein has three structurally homologous domains (domains I, II, and III), nearly all of which reside in an alpha-helical conformation and are highly stabilized by 17 disulfide bridges. In certain preferred embodiments, the Affimer polypeptide can be an albumin fusion protein comprising one or more Affimer polypeptide sequences and, to the extent desired in the fusion protein, the sequence of mature human serum albumin (SEQ ID NO: 17), or a variant or fragment thereof that maintains the PK and / or biodistribution properties of mature albumin. DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 17)

[0438] The albumin sequence can be separated from the Affimer polypeptide sequence or other adjacent sequences in the Affimer polypeptide by the use of linker sequences as described above.

[0439] Unless otherwise indicated, references herein to "albumin" or "mature albumin" are intended to refer to HSA. However, it is noted that full-length HSA has an 18-amino acid (MKWVTFISLLFLFSSAYS) signal peptide followed by a 6-amino acid (RGVFRR) prodomain; this 24-amino acid residue peptide is sometimes referred to as the preprodomain. The HSA preprodomain within a recombinant protein coding sequence can be used to express and secrete Affimer-HSA fusion proteins. Alternatively, Affimer-HSA fusions can be expressed and secreted by including other secretory signal sequences, such as those described above.

[0440] In an alternative embodiment, rather than being provided as part of a fusion protein with an Affimer polypeptide, the serum albumin polypeptide may be covalently coupled to the Affimer-containing polypeptide by a bond other than a backbone amide bond, e.g., cross-linked by chemical conjugation between the amino acid side chains of the albumin polypeptide and the Affimer-containing polypeptide.

[0441] (iii) albumin-binding domain In certain embodiments, the Affimer polypeptide may include a serum-binding moiety as part of a fusion protein (even a polypeptide) with the Affimer polypeptide sequence, or as a chemically conjugated moiety via a site other than that which is part of a continuous polypeptide chain.

[0442] In certain embodiments, the serum-binding polypeptide is an albumin-binding moiety. Albumin contains multiple hydrophobic binding pockets and naturally functions as a transporter of various different ligands, such as fatty acids and steroids, and as a transporter of different drugs. Furthermore, the surface of albumin is negatively charged, making it highly water-soluble.

[0443] The term "albumin binding moiety" as used herein refers to any chemical group that can bind to albumin, i.e., has albumin binding affinity. Albumin binds to endogenous ligands such as fatty acids, but also interacts with exogenous ligands such as warfarin, penicillin, and diazepam. Because the binding of these drugs to albumin is reversible, albumin-drug complexes serve as drug reservoirs that can improve the biodistribution and bioavailability of drugs. The incorporation of components that mimic endogenous albumin binding ligands, such as fatty acids, has been used to enhance albumin association and increase drug efficacy.

[0444] In certain embodiments, a chemical modification method that can be applied to the production of a subject Affimer polypeptide to extend protein half-life is lipidation, which involves covalently attaching a fatty acid to a peptide side chain. Lipidation, originally conceived and developed as a method to extend the half-life of insulin, shares the same basic mechanism of half-life extension as PEGylation: increasing the hydrodynamic radius and reducing renal filtration. However, the lipid moiety itself is relatively small, and the effect is indirectly mediated by the non-covalent binding of the lipid moiety to circulating albumin. One significance of lipidation is that it reduces the water solubility of the peptide, but this can be modulated by modifying the linker between the peptide and the fatty acid, for example, by using glutamic acid or mini-PEG in the linker. Linker modification and alteration of the lipid moiety can affect self-aggregation, which can contribute to half-life extension by slowing biodistribution independent of albumin. See, for example, Jonassen et al. (2012) Pharm Res. 29(8):2104-14.

[0445] Other examples of albumin binding moieties for use in generating certain Affimer polypeptides include albumin-binding (PKE2) Adnectins (see WO2011140086 "Serum Albumin Binding Molecules," WO2015143199 "Serum albumin-binding Fibronectin Type III Domains," and WO2017053617 "Fast-off rate serum albumin binding fibronectin type III domains"), albumin binding domain 3 (ABD3) of protein G of Streptococcus strain G148, and albumin binding domain antibody ("AlbudAb") of ATN-103 (ozoralizumab) GSK2374697 or albumin-binding nanobody moieties.

[0446] (iv) PEGylation, XTEN, PAS and other polymers A wide variety of large polymers and other molecules can be linked to the Affimer-containing polypeptides of the present disclosure to modulate and / or confer new biological properties on the resulting Affimer polypeptide. These large polymers can be linked to the Affimer-containing polypeptides through naturally encoded amino acids, non-naturally encoded amino acids, or any functional substituent of a natural or unnatural amino acid, or any substituent or functional group added to a natural or unnatural amino acid. The molecular weight of the polymer can vary widely, including, but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer is 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 1 The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including, but not limited to, 0,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 and about 40,000 Da.

[0447] For this purpose, various methods have been developed, including PEGylation, polysialylation, hydroxyethyl esterification, glycosylation, or recombinant PEG analogs fused to flexible hydrophilic amino acid chains (500-600 amino acids) (see Chapman, (2002) Adv Drug Deliv Rev. 54, 531-545; Schlapschy et al., (2007) Prot Eng Des Sel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246).

[0448] Examples of polymers include polyalkyl ethers and alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene / propylene glycol, and methoxy- or ethoxy-capped analogs thereof, particularly polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidone; polyvinyl alkyl ethers; polyoxazolines, polyalkyloxazolines, and polyhydroxyalkyloxazolines; polyacrylamides, polyalkylacrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and its derivatives); polyhydroxyalkylacrylates; polysialic acid and its analogs; hydrophilic peptide sequences; polysaccharides and their derivatives (including dextran and dextran derivatives, e.g., carboxymethyl dextran, dextran sulfate, aminodextran); cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl cellulose, hydroxypropyl ... Examples of suitable polysaccharides include, but are not limited to, cellulose; chitin and its derivatives, such as chitosan, succinyl chitosan, carboxymethyl chitin, and carboxymethyl chitosan; hyaluronic acid and its derivatives; starch; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyamino acids and their derivatives, such as polyglutamic acid, polylysine, polyaspartic acid, and polyaspartamide; maleic anhydride copolymers, such as styrene-maleic anhydride copolymer and divinyl ethyl ether-maleic anhydride copolymer; polyvinyl alcohol; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives of the foregoing.

[0449] The polymer selected may be water-soluble so that the Affimer polypeptide to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The water-soluble polymer may be of any structural form, including, but not limited to, linear, forked, or branched. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), although other water-soluble polymers may also be used. PEG is used as an example to illustrate certain embodiments of the present disclosure. For therapeutic uses of Affimer polypeptides, the polymer may be pharmaceutically acceptable.

[0450] The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of its size or modification at its termini, and when linked to an Affiner-containing polypeptide, has the formula: XO-(CH2CH2O) n -CH2CH2- or XO-(CH2CH2O) n - where n is 2 to 10,000, and X is H or a terminal modification, including, but not limited to, C alkyl, a protecting group, or a terminal functional group. In some cases, PEG used in polypeptides of the present disclosure terminates at one end with hydroxy or methoxy, i.e., X is H or CH ("methoxy PEG").

[0451] The other end of the PEG, indicated by the terminal "-" in the formula above, may be attached to the Affimer-containing polypeptide via a naturally occurring or non-naturally encoded amino acid. For example, the linkage may be to an amine group of the polypeptide (including, but not limited to, the epsilon amine of lysine, or the N-terminus) via an amide, carbamate, or urea bond. Alternatively, the polymer may be linked to a thiol group (including, but not limited to, the thiol group of cysteine) via a maleimide bond; this requires the conversion of a residue in the Affimer sequence to cysteine ​​for attachment to the Affimer polypeptide sequence itself.

[0452] The number of water-soluble polymers linked to an Affimer-containing polypeptide (i.e., the degree of PEGylation or glycosylation) can be adjusted to result in an alteration (including, but not limited to, an increase or decrease) in the pharmacological, pharmacokinetic, or pharmacodynamic properties, such as the in vivo half-life, of the resulting Affimer polypeptide. In some embodiments, the half-life of the resulting Affimer polypeptide is extended by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold compared to the unmodified polypeptide.

[0453] Another variation of polymer systems useful for modifying the PK or other biological properties of the resulting Affimer polypeptide is the use of amorphous hydrophilic amino acid polymers, which are functional analogs of PEG, particularly as part of a fusion with the Affimer polypeptide sequence. The inherent biodegradability of the polypeptide platform makes it attractive as a potentially better alternative to PEG. Another advantage is the more precise molecular structure of the recombinant molecule, as opposed to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, which require maintaining the three-dimensional folding of the fusion partner, recombinant fusions with amorphous partners can often be subjected to higher temperatures or more stringent conditions, such as HPLC purification.

[0454] A more advanced member of this class of polypeptides, called XTEN (Amunix), is 864 amino acids long and composed of six amino acids (A, E, G, P, S, and T). See Schellenberger et al. "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner," 2009 Nat Biotechnol. 27(12):1186-90. This is made possible by the biodegradable nature of the polymer, resulting in a much greater half-life extension than the commonly used 40 kDa PEG, concomitantly resulting in a much greater half-life extension. Fusion of XTEN to an Affimer-containing polypeptide should result in a 60- to 130-fold increase in the half-life of the final Affimer polypeptide compared to the unmodified peptide.

[0455] A second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH). Schlapschy et al. "PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins" 2013 Protein Eng Des Sel. 26(8):489-501. Random coil polymers are composed of a much more restricted set of only three small, uncharged amino acids: proline, alanine, and serine. Similar to Fc, HAS, and XTEN, the PAS modification can be genetically encoded using an Affimer polypeptide sequence to produce an in-line fusion protein when expressed.

[0456] b. Multispecific fusion proteins In certain embodiments, the Affimer polypeptide is a multispecific and / or multivalent polypeptide, comprising, for example, a first Affimer polypeptide that binds to a first target and at least one additional binding domain (which may be an entirely different molecule (bispecific), or the same molecule type at the same site (multivalent), or the same molecule but at different sites (biparatopic or multiparatopic)) that binds to a second target different from the first target. The additional binding domain may be a polypeptide sequence, illustratively selected from among a second Affimer polypeptide sequence (which may be the same or different from the first Affimer polypeptide sequence), an antibody or fragment thereof or other antigen-binding polypeptide, a ligand-binding portion of a receptor (e.g., a receptor-trap polypeptide), a receptor-binding ligand (e.g., a cytokine, growth factor, or the like), a modified T-cell receptor, an enzyme or catalytic fragment thereof, or other polypeptide sequence that confers some function.

[0457] In certain embodiments, an Affimer polypeptide comprises one or more antigen-binding sites from an antibody. The resulting taAffimer polypeptide contains both Affimer sequences and sequences for antibody antigen-binding sites (e.g., in the case of scFVs), and can be a single chain or a multimeric protein complex, such as in an antibody assembled with heavy and / or light chains to which Affimer sequences are also fused.

[0458] In some embodiments of multispecific Affimer polypeptides, including full-length immunoglobulins, fusion of the Affimer polypeptide sequence to an antibody will disrupt the Fc function of the Fc region of the immunoglobulin. For example, in certain embodiments, the Affimer polypeptide will be able to bind to an Fc receptor on an Fc receptor-positive cell via the Fc portion. In some further embodiments, the Affimer polypeptide can activate an Fc receptor-positive cell upon binding to the Fc receptor-positive cell, thereby initiating or increasing the expression of cytokines and / or costimulatory antigens. Furthermore, the Affimer polypeptide can transfer at least a second activation signal to the T cell via a costimulatory antigen and / or cytokine, which is necessary for physiological activation of the T cell.

[0459] In some embodiments, due to the binding of the Fc portion to other cells expressing Fc receptors present on the surface of effector cells from the immune system, such as immune cells, hepatocytes, and endothelial cells, the Affimer polypeptides can have antibody-dependent cellular cytotoxicity (ADCC) function, a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound by antibodies, thus inducing tumor cell death by ADCC. In some further embodiments, the Affimer polypeptides can exhibit ADCC function.

[0460] In addition to the Fc-mediated cytotoxicity described above, Fc fusions can contribute to maintaining serum levels of Affimer polypeptides, which is essential for their stability and persistence in the body. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the Affimer polypeptides can be internalized and recycled back into the bloodstream, thereby increasing their half-life in the body.

[0461] Exemplary targets for additional Affimer polypeptides include, by way of illustration only, but are not limited to, another immune checkpoint protein, and an immune costimulatory receptor (particularly where the additional Affimer recognizes a costimulatory receptor), a receptor, a cytokine, a growth factor, or a tumor-associated antigen.

[0462] C. conjugate A subject Affimer polypeptide can also contain one or more functional moieties intended to confer detectability or additional pharmacological activity to the Affimer polypeptide. A functional moiety for detection is one that can be used to detect the association of an Affimer polypeptide with a cell or tissue (e.g., tumor cell) in vivo. A pharmacologically active functional moiety is an agent intended to be delivered to a tissue expressing the target of the Affimer polypeptide and, in doing so, to have a pharmacological effect on the target tissue or cell.

[0463] The present disclosure relates to Affimer polypeptides comprising conjugates of substances and functional moieties having a wide variety of functional groups, substituents, or moieties, where these functional moieties include labels; dyes; immunoadhesion molecules; radionuclides; cytotoxic compounds; drugs; affinity labels; photoaffinity labels; reactive compounds; resins; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; metal chelators; cofactors; fatty acids; carbohydrates; polynucleotides; DNA; RNA; antisense polynucleotides; saccharides; water-soluble dendrimers; cyclodextrins; inhibitory ribonucleic acids; biomaterials; nanoparticles; spin labels; fluorophores, metal-containing moieties; radioactive moieties; novel functional groups; groups that interact covalently or non-covalently with other molecules; photocaged moieties; Affimer polypeptides are provided, including, but not limited to, actinic excitable moieties; photoisomerizable moieties; biotin; derivatives of biotin; biotin analogs; moieties incorporating heavy atoms; chemically cleavable groups; photocleavable groups; extended side chains; carbon-linked sugars; redox-active agents; aminothioacids; toxic moieties; isotopically labeled moieties; biophysical probes; phosphorescent groups; chemiluminescent groups; electron-dense groups; magnetic groups; intercalating groups; chromophores; energy transfer agents; biologically active agents; detectable labels; small molecules; quantum dots; nanotransmitters; radionucleotides; radiotransmitters; neutron capture agents; or any combination of the above; or any other desired compound or substance.

[0464] (i) Labels and Detectable Moieties When the moiety is a detectable label, it can be a fluorescent label, a radioactive label, an enzymatic label, or any other label known to those skilled in the art. In certain embodiments, the functional moiety is a detectable label that can be included as part of a conjugate to make certain Affimer polypeptides suitable for medical imaging. "Medical imaging" refers to any technique used to visualize internal regions of the human or animal body for the purposes of diagnosis, research, or therapeutic treatment. For example, Affimer polypeptides can be detected (and quantified) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT) (CT scan), nuclear imaging, positron emission tomography (PET) contrast agents containing metals, optical imaging (e.g., fluorescence imaging, including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or a combination thereof. The functional moiety can also be a contrast agent for X-ray imaging. Agents useful for enhancing such procedures are substances that allow visualization of specific locations, organs, or disease sites within the body and / or lead to some improvement in the quality of images produced by the imaging procedure, resulting in improved and easier interpretation of those images. Such agents are referred to herein as contrast agents, and their use facilitates discrimination of different parts of an image by increasing the "contrast" between different regions of the image. Thus, the term "contrast agent" encompasses agents used to improve image quality, as well as agents that are necessary prior to the production of an image (e.g., as in nuclear imaging), although they may be produced in the absence of such agents (e.g., as in MRI).

[0465] In certain preferred embodiments, the detectable label comprises a chelating moiety for chelating a metal, e.g., a chelator for a radiometal or a paramagnetic ion. In certain preferred embodiments, the detectable label is a chelator for a radionuclide useful in radiotherapy or imaging procedures. Radionuclides useful within the present invention include gamma emitters, positron emitters, Auger electron emitters, X-ray emitters, and fluorescent emitters, with beta or alpha emitters being preferred for therapeutic use. Examples of radionuclides useful as toxins in radiation therapy include 43K, 47Sc, 51Cr, 57Co, 58Co, 59Fe, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 81Rb, 90Y, 97Ru, 99mTc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 111Ag, 111In, 113In, These include Sb, Sn, I, I, Cs, Ba, Cs, I, Cs, Pr, Sm, Tb, Ho, Eu, Lu, Re, Re, Re, Os, Pt, Ir, Hg, Au, Pb, At, Pb, Bi, and Bi. Conditions for chelating agents to coordinate to metals are described, for example, by Gansow et al. in U.S. Pat. Nos. 4,831,175, 4,454,106, and 4,472,509. Examples of chelating agents include, by way of illustration only, 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid (DOTA), and 1,4,8,11-tetraazacyclotetradecane-N,N',N",N'''-tetraacetic acid (TETA).

[0466] Other detectable isotopes that can be incorporated directly into amino acid residues of the Affimer polypeptide, or which do not otherwise require a chelating agent, include 3H, 14C, 32P, 35S, and 36Cl.

[0467] Paramagnetic ions useful in diagnostic procedures can also be administered. Examples of paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), or combinations of these paramagnetic ions.

[0468] Examples of fluorescent labels include, but are not limited to, organic dyes (e.g., cyanine, fluorescein, rhodamine, Alexa Fluor, Dylight fluor, ATTO Dye, BODIPY dyes, etc.), biological fluorophores (e.g., green fluorescent protein (GFP), R-phycoerythrin, etc.), and quantum dots.

[0469] Non-limiting examples of fluorescent compounds that can be used in the present invention include Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as CY3++), Texas Red, LightCycler-Red 640, LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), rhodamine derivative JA133, Alexa Fluor dyes (e.g., Alexa Fluor 488, Alexa Fluor 500), and the like. Fluorescent compounds that can be used include 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes such as europium. Fluorescent compounds that can be used include fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent proteins and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent proteins and derivatives (CFP, ECFP, Cerulean, CyPet), and yellow fluorescent proteins and derivatives (YFP, Citrine, Venus, YPet). International Publication No. 2008142571, International Publication No. 2009056282, International Publication No. 9922026.

[0470] Examples of enzymatic labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and -galactosidase.

[0471] Another well-known label is biotin. Biotin labels typically consist of a biotinyl group, a spacer arm, and a reactive group that participates in binding to a target functional group on a protein. Biotin can be useful for binding labeled proteins to other moieties, including avidin moieties.

[0472] (ii) Affimer-drug conjugates In certain embodiments, an Affimer polypeptide includes one or more therapeutic agents, for example, to form an Affimer-drug conjugate. As used herein, the term "therapeutic agent" refers to a substance that can be used to cure, mitigate, treat, or prevent disease in a human or another animal. Such therapeutic agents include substances recognized in the Official United States Pharmacopoeia, the Official United States Homeopathic Pharmacopoeia, the Official National Formulary, or any supplements thereof, including, but not limited to, small molecules, nucleotides, oligopeptides, polypeptides, and the like. Therapeutic agents that can be conjugated to Affimer-containing polypeptides include, but are not limited to, cytotoxic agents, antimetabolites, alkylating agents, antibiotics, growth factors, cytokines, antiangiogenic agents, antimitotic agents, toxins, apoptotic agents, or the like, such as, by way of example, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, vinca alkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers, and chemotherapy combination treatments.

[0473] Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as mechlorethamine, cyclophosphamide (ifosfamide, trofosfamide), chlorambucil (melphalan, prednimustine), bendamustine, uramustine, and estramustine; nitrosoureas, such as carmustine (BCNU), lomustine (semustine), fotemustine, nimustine, ranimustine, and streptozocin; alkylsulfonic acids, such as busulfan (mannosulfan, treosulfan); aziridines, such as carboquone, thioTEPA, triaziquone, triethylenemelamine; hydrazine (procarbazine); triazenes, such as dacarbazine and temozolomide; altretamine, and mitobronitol.

[0474] Non-limiting examples of topoisomerase I inhibitors include camptothecin derivatives, including CPT-11 (irinotecan), SN-38, APC, NPC, camptothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lutotecan, rubidecan, siratecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT, as described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and U.S. Patent Application Publication No. 200510250854; Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800; phenanthroline derivatives, including benzo[i]phenanthridine, nitidine, and fagaronine, as described in Makhey et al. (2003) Bioorg. Med. Chem. 11 (8): 1809-1820; terbenzimidazole and its derivatives, as described in Xu (1998) Biochemistry 37(10):3558-3566; and Foglesong et al. (1992) Cancer Chemother. Pharmacol. 30(2):123-25, Crow et al. (1994) J. Med. Chem. 37(19):3191-3194, and Crespi et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8, including anthracycline derivatives, including doxorubicin, daunorubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, etoposide and teniposide.Dual topoisomerase I and II inhibitors include, but are not limited to, saintopine and other naphthacenediones, DACA and other acridine-4-carboxamides, intoplicin and other benzopyridoindoles, TAS-103 and other 7H-indeno[2,1-c]quinolin-7-ones, pyrazoloacridines, XR11576 and other benzophenazines, XR5944 and other dimeric compounds, 7-oxo-7H-dibenz[f,ij]isoquinolines and 7-oxo-7H-benzo[e]pyrimidines, and anthracenyl-amino acid conjugates, as described in Denny and Baguley (2003) Curr. Top. Med. Chem. 3(3):339-353. Some drugs, such as, but not limited to, the anthracyclines (aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin) and the anthracenediones (mitoxantrone and pixantrone), inhibit topoisomerase II and have DNA intercalating activity.

[0475] Examples of endoplasmic reticulum stress inducers include, but are not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (i.e., Velcade (bortezomib)).

[0476] Non-limiting examples of platinum-based compounds include carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate, satraplatin, aroplatin, lobaplatin, and JM-216 (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237, and generally CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in the Series Basic and Clinical Oncology, Angioli et al. Eds., 2004).

[0477] Non-limiting examples of antimetabolites include those based on folic acid, i.e., dihydrofolate reductase inhibitors, such as aminopterin, methotrexate, and pemetrexed; thymidylate synthase inhibitors, such as raltitrexed, pemetrexed; those based on purine, i.e., adenosine deaminase inhibitors, such as pentostatin, thiopurines, such as thioguanine and mercaptopurine, halogenated / ribonucleotide reductase inhibitors, such as cladribine, clofarabine, fludarabine, or guanine / guanosine:thiopurines, such as thioguanine; or those based on pyrimidine, i.e., cytosine / cytidine:hypomethylating agents, such as azacitidine and decitabine, DNA polymerase inhibitors, such as cytarabine, ribonucleotide reductase inhibitors, such as gemcitabine, or thymine / thymidine:thymidylate synthase inhibitors, such as fluorouracil (5-FU). Equivalents of 5-FU include its prodrugs, analogs and derivatives, such as 5'-deoxy-5-fluorouridine (doxifluridine), 1-tetrahydrofuranyl-5-fluorouracil (Ftorfur), capecitabine (Xeloda), SI (MBMS-247616, which consists of tegafur and two modulators, 5-chloro-2,4-dihydropyridine and potassium oxonate), raltitrexed (Tomudex), nolatrexed (Timitac, AG337), LY231514 and ZD9331, as described, for example, in Papamicheal (1999) The Oncologist 4:478-487.

[0478] Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, vinflunine, vindesine, and vinorelbine.

[0479] Examples of taxanes include, but are not limited to, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel.An example of an epothilone is ixabepilone.

[0480] Examples of enzyme inhibitors include, but are not limited to, farnesyltransferase inhibitors (tipifarnib); CDK inhibitors (alvocidib, seliciclib); proteasome inhibitors (bortezomib); phosphodiesterase inhibitors (anagrelide; rolipram); IMP dehydrogenase inhibitors (tiazofurin); and lipoxygenase inhibitors (masoprocol). Examples of receptor antagonists include, but are not limited to, ERAs (atrasentan); retinoid X receptor (bexarotene); and sex steroids (testolactone).

[0481] Examples of therapeutic antibodies include, but are not limited to, anti-HER1 / EGFR antibodies (cetuximab, panitumumab), anti-HER2 / neu (erbB2) receptor (trastuzumab), anti-EpCAM antibodies (catumaxomab, edrecolomab), anti-VEGF-A antibodies (bevacizumab), anti-CD20 antibodies (rituximab, tositumomab, ibritumomab), anti-CD52 antibodies (alemtuzumab), and anti-CD33 antibodies (gemtuzumab). See U.S. Patent Nos. 5,776,427 and 7,601,355.

[0482] Examples of tyrosine kinase inhibitors include, but are not limited to, inhibitors against ErbB: HER1 / EGFR (erlotinib, gefitinib, lapatinib, vandetanib, sunitinib, neratinib); HER2 / neu (lapatinib, neratinib); RTK class III: C-kit (axitinib, sunitinib, sorafenib), FLT3 (lestaurtinib), PDGFR (axitinib, sunitinib, sorafenib); and VEGFR (vandetanib, semaxanib, cediranib, axitinib, sorafenib); bcr-abl (imatinib, nilotinib, dasatinib); Src (bosutinib) and Janus kinase 2 (lestaurtinib).

[0483] Chemotherapeutic agents that can be conjugated to the present Affimer-containing polypeptides also include amsacrine, trabectedin, retinoids (alitretinoin, tretinoin), arsenic trioxide, asparagine depleting agents (asparaginase / pegaspargase), celecoxib, demecolcine, elexromol, elsamitrucin, etoglucide, lonidamine, lucantone, mitoguazone, mitotane, oblimersen, temsirolimus, and vorinostat.

[0484] Examples of specific therapeutic agents that can be linked, ligated, or associated with the Affimer-containing polypeptides of the present invention include flomoxef; fortimicin; gentamicin; glucosulfone; solasulfone; gramicidin S; gramicidin; grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycin; flomoxef; fortimicin; gentamicin; glucosulfone; solasulfone; gramicidin S; gramicidin; grepafloxacin; guamecycline; hetacillin; isepamicin Isin; Josamycin; Kanamycin; Bacitracin; Bambermycin; Biapenem; Brodimoprim; Butirosin; Capreomycin; Carbenicillin; Carbomycin; Carumonam; Cefadroxil; Cefamandole; Cefatrizine; Cefbuperazone; Cefclidin; Cefdinir; Cefditoren; Cefepime; Cefetamet; Cefixime; Cefmenoxime; Cefminox; Cladribine; Apalcillin; Apicycline; Apramycin; Arbekacin; Aspoxicillin; Azidamphenicol; Aztreonam; Cefodizime; Cef Onicid; Cefoperazone; Ceforanide; Cefotaxime; Cefotetan; Cefotiam; Cefozopran; Cefpimizole; Cefpiramide; Cefpirome; Cefprozil; Cefroxadine; Cefteram; Ceftibuten; Cefuzonam; Cephalexin; Cephaloglycin; Cephalosporin C; Cephradine; Chloramphenicol; Chlortetracycline; Clinafloxacin; Clindamycin; Clomocycline; Colistin; Cyclacillin; Dapsone; Demeclocycline; Diatimosulfone; Dibekacin; Dihydrostreptomycin; 6-Methionine Lucaptopurine; Thioguanine; Capecitabine; Docetaxel; Etoposide; Gemcitabine; Topotecan; Vinorelbine; Vincristine; Vinblastine; Teniposide; Melphalan; Methotrexate; 2-p-Sulfanylanilinoethanol; 4,4'-Sulfinyldianiline; 4-Sulfanilamidosalicylic acid; Butorphanol; Nalbuphine; Streptozocin; Doxorubicin; Daunorubicin; Plicamycin; Idarubicin; Mitomycin C; Pentostatin; Mitoxantrone; Cytarabine; Fludarabine phosphate;Butorphanol; Nalbuphine; Streptozocin; Doxorubicin; Daunorubicin; Plicamycin; Idarubicin; Mitomycin C; Pentostatin; Mitoxantrone; Cytarabine; Fludarabine phosphate; Acediasulfone; Acetosulfone; Amikacin; Amphotericin B; Ampicillin; Atorvastatin; Enalapril; Ranitidine; Ciprofloxacin; Pravastatin; Clarithromycin; Cyclosporine; Famotidine; Leuprolide; Acyclovir; Paclitaxel; Azithromycin; Lamivudine; Budesonide ;Albuterol;Indinavir;Metformin;Alendronate;Nizatidine;Zidovudine;Carboplatin;Metoprolol;Amoxicillin;Diclofenac;Lisinopril;Ceftriaxone;Captopril;Salmeterol;Xinafoate;Imipenem;Cilastatin;Benazepril;Cefaclor;Cefazidime;Morphine;Dopamine;Bialamicol;Fluvastatin;Phenamidine;Podophyllinic acid 2-ethylhydrazine;Acriflavine;Chlorazodine;Arsphenamine;Amicarbilide;Ami Quinuride; Quinapril; Oxymorphone; Buprenorphine; Floxuridine; Dirithromycin; Doxycycline; Enoxacin; Enviomycin; Epicillin; Erythromycin; Leucomycin; Lincomycin; Lomefloxacin; Lucensomycin; Lymecycline; Meclocycline; Meropenem; Methacycline; Micronomycin; Midecamycin; Minocycline; Moxalactam; Mupirocin; Nadifloxacin; Natamycin; Neomycin; Netilmicin; Norfloxacin; Oleandomycin; Oxytetracycline Cyclins; p-sulfanylbenzylamine; panipenem; paromomycin; pazufloxacin; penicillin N; pipacycline; pipemidic acid; polymyxins; primycin; quinacillin; ribostamycin; rifamide; rifampin; rifamycin SV; rifapentine; rifaximin; ristocetin; ritipenem; rokitamycin; rolitetracycline; rosaramycin; roxithromycin; salazosulfadimidine; sancycline; sisomicin; sparfloxacin; spectinomycin; spiramycin; streptomycin;Succisulfone; Sulfachrysoidine; Sulfaloxic acid; Sulfamidochrysoidine; Sulfanilic acid; Sulfoxone; Teicoplanin; Temafloxacin; Temocillin; Tetroxoprim; Thiamphenicol; Thiazole sulfone; Thiostrepton; Ticarcillin; Tigemonam; Tobramycin; Tosufloxacin; Trimethoprim; Trospectomycin; Trovafloxacin; Tuberactinomycin; Vancomycin; Azaserine; Candicin; Chlorphenesin; Dermostatin; Philippine; Fungichromin; Mepartricin; Nith Tatin; Oligomycin; Perimycin A; Tubercidin; 6-Azauridine; 6-Diazo-5-oxo-L-norleucine; Aclacinomycin; Ancitabine; Anthramycin; Azacitidine; Azaserine; Bleomycin; Ethyl bismuth acetate; Ethylidene dicumarol; Iloprost; Lamifiban; Taprosten; Thiochromarol; Tirofiban; Amiprilose; Bucillamine; Gluperimus; Gentisic acid; Glucametacin; Glycol salicylate; Meclofenamic acid; Mefenamic acid; Mesalamine; Niflumic acid; Olsalazine; Oxacetoacetate Prole; S-enosylmethionine; salicylic acid; salsalate; sulfasalazine; tolfenamic acid; carubicin; carzinophilin A; chlorozotocin; chromomycin; denopterin; doxifluridine; edatrexate; eflornithine; elliptinium; enocitabine; epirubicin; mannomustine; menogaril; mitobronitol; mitolactol; mopidamol; mycophenolic acid; nogalamycin; olivomycin; peplomycin; pirarubicin; piritrexim; prednimustine; Procarbazine; Pteropterin; Puromycin; Ranimustine; Streptonigrin; Thiamiprine; Mycophenolic acid; Procodazole; Romurtide; Sirolimus (rapamycin); Tacrolimus; Butethamine; Fenalcomine; Hydroxytetracaine; Nepine; Orthocaine; Piridocaine; Salicylic alcohol; 3-amino-4-hydroxybutyric acid; Aceclofenac; Alminoprofen; Amfenac; Bromfenac; Bromosaligenin; Bumadizone; Carprofen; Diclofenac; Diflunisal; Ditazol; Enfenamic acid;Etodolac; etofenamate; fendosal; feprazinol; flufenamic acid; tomudex (N-[[5-[[(1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]methylamino]-2-thienyl]carbonyl]-L-glutamic acid), trimetrexate, tubercidin, ubenimex, vindesine, zorubicin; argatroban; coumetalol or dicoumarol;

[0485] In certain embodiments, the Affimer polypeptide comprises a conjugated cytotoxic factor, such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin protein, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.

[0486] Any method known in the art for conjugating antibodies and other proteins can be used to produce the conjugates of the invention, including those described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating peptides, polypeptides, and organic and inorganic moieties to antibodies and other proteins are conventional and well known in the art and are readily adapted to produce these versions of the subject Affiner polypeptides.

[0487] When the conjugated moiety is a peptide or polypeptide, the moiety can be chemically crosslinked to the Affimer-containing polypeptide or can be included as part of a fusion protein with the Affimer-containing polypeptide. An illustrative example would be a diphtheria toxin-Affimer fusion protein. For non-peptide entities, attachment to the Affimer-containing polypeptide will generally be by chemical conjugation to the Affimer-containing polypeptide (e.g., via a functional group on an amino acid side chain of the polypeptide or a carboxyl group at the C-terminus or an amino group at the N-terminus). In certain embodiments, the conjugated moiety, whether as a fusion protein or a chemically crosslinked moiety, will contain one or more sites that can be enzymatically cleaved or otherwise sensitive to environmental conditions (e.g., pH) that allow the conjugated moiety to be released from the Affimer-containing polypeptide, such as the environmental conditions in a tumor or other diseased tissue (or the tissue to be protected, if the conjugated moiety functions to protect healthy tissue).

[0488] III. Expression Methods and Systems The recombinant Affimer-containing proteins described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding polypeptide sequences and expressing those sequences in a suitable host. For those recombinant Affimer polypeptides that contain further modifications, such as chemical modification or conjugation, the recombinant Affimer polypeptides can be further manipulated chemically or enzymatically after isolation from host cells or chemical synthesis.

[0489] The present invention includes recombinant methods and nucleic acids for recombinant expression of a recombinant Affimer polypeptide of the present invention, comprising the steps of (i) introducing into a host cell a polynucleotide encoding the amino acid sequence of the Affimer polypeptide, e.g., in a vector and / or operably linked to a promoter, (ii) culturing the host cell (e.g., a eukaryotic or prokaryotic cell) under conditions suitable for expression of the polynucleotide, and (iii) optionally isolating the Affimer polypeptide from the host cell and / or from the medium in which the host cell is grown. See, e.g., WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0490] In some embodiments, a DNA sequence encoding a desired recombinant Affimer polypeptide can be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the codon selection that is advantageous in the host cell in which the desired recombinant polypeptide will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding a desired isolated polypeptide. For example, a complete amino acid sequence can be used to construct a reverse-translated gene. Additionally, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, multiple small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0491] Once a nucleic acid sequence encoding a recombinant Affimer polypeptide of the present invention is obtained, vectors for producing the recombinant Affimer polypeptide can be produced by recombinant DNA technology using techniques well known in the art. Methods well known to those skilled in the art can be used to construct expression vectors containing the recombinant Affimer polypeptide coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al., 1990, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al. eds., 1998, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY.)

[0492] Expression vectors containing the nucleotide sequence of a recombinant Affimer polypeptide can be transferred into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the recombinant Affimer polypeptide of the invention. In certain embodiments, expression of the recombinant Affimer polypeptide is regulated by a constitutive, inducible, or tissue-specific promoter.

[0493] Expression vectors may contain an origin of replication, e.g., an origin of replication, which may be selected based on the type of host cell to be used for expression. By way of example, the origin of replication from the plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass.) is useful for most Gram-negative bacteria, while various origins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomavirus (e.g., HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, those from SV40 are often used because they contain the early promoter).

[0494] Vectors may also contain one or more selectable marker genes, e.g., genetic elements encoding proteins necessary for the production and growth of host cells grown in selective culture media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to prokaryotic host cells, (b) complement auxotrophic deficiencies of the cells, or (c) supply essential nutrients unavailable from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. The neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells. Other selection genes can be used to amplify the expressed gene. Amplification is the process by which a gene with greater demand for production of a protein essential for growth is tandemly repeated in the chromosome of successive generations of recombinant cells. Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Mammalian cell transformants are placed under selection pressure where only the transformants are uniquely adapted to survive due to the marker present in the vector. Selection pressure is exerted by culturing the transformed cells under conditions that allow for the successful alteration of the concentration of the selective agent in the medium, thereby amplifying both the selection gene and the DNA encoding the recombinant Affimer polypeptide. As a result, increased amounts of the recombinant Affimer polypeptide are synthesized from the amplified DNA.

[0495] The vector may also contain one or more ribosome binding sites that result in transcription into mRNA containing the coding sequence of the recombinant Affimer polypeptide. For example, such sites are characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' of the promoter and 5' of the coding sequence of the polypeptide to be expressed. Shine-Dalgarno sequences vary but are usually polypurines (with a high AG content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using the methods described above and used in prokaryotic vectors.

[0496] Expression vectors will usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid molecule encoding the recombinant Affimer polypeptide. Depending on the host cell used for expression and the desired yield, either the native promoter or a heterologous promoter can be used.

[0497] Promoters for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase; tryptophan (trp) promoter systems; and hybrid promoters, such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences are publicly available, and they can be ligated to the desired nucleic acid sequence, using linkers or adapters, as needed, to provide restriction sites.

[0498] Promoters for use with yeast hosts are also known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably, Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and the actin promoter.

[0499] Additional promoters that can be used to express the selective binding agents of the invention include the SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci. USA 78:1444-5); the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296; 39-42, 1982); and promoters of prokaryotic expression vectors, such as the beta-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA, 75; 3727-3731, 1978); or the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad. Sci. USA, 80: 21-5). Also of interest are the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al. (1984), Cell 38: 639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald (1987), Hepatology 7: 425-515); the insulin gene control region, which is active in pancreatic beta cells (Hanahan (1985), Nature 315: 115-22); and the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al. (1984), Cell 38; 647-58; Adames et al. (1985), Nature 318; 533-8; Alexander et al. (1987), Mol. Cell. Biol.7: 1436-44); mouse mammary tumor virus control region, which is active in testis, breast, lymphoid, and mast cells (Leder et al. (1986), Cell 45: 485-95); albumin gene control region, which is active in liver (Pinkert et al. (1987), Genes and Devel. 1: 268-76); alpha-fetoprotein gene control region, which is active in liver (Krumlauf et al. (1985), Mol. Cell. Biol. 5: 1639-48; Hammer et al. (1987), Science, 235: 53-8); alpha-1-antitrypsin gene control region, which is active in liver (Kelsey et al. (1987), Genes and Devel. 1: 161-71); beta-globin gene control region, which is active in myeloid cells (Mogram et al., Nature, 315 338-340, 1985; Kollias et al. (1986), Cell 46: 89-94); the myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al. (1987), Cell, 48: 703-12); the myosin light chain-2 gene control region, which is active in skeletal muscle (Sani (1985), Nature, 314: 283-6); and the gonadotropin-releasing hormone gene control region, which is active in the hypothalamus (Mason et al. (1986), Science 234: 1372-8).

[0500] An enhancer sequence can be inserted into the vector to increase transcription in eukaryotic host cells. Several enhancer sequences are known that can be obtained from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from viruses are usually used. SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancing elements for the activation of eukaryotic promoters.

[0501] Although the enhancer may be spliced ​​into the vector at a position 5' or 3' to the polypeptide coding region, it is usually located at a site 5' from the promoter.

[0502] Vectors for expressing nucleic acids include those compatible with bacterial, insect, and mammalian host cells, such as pCRII, pCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. WO 90 / 14363), and pFastBacDual (Gibco / BRL, Grand Island, NY), among others.

[0503] Additional possible vectors include, but are not limited to, cosmids, plasmids, or modified viruses, but the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to, plasmids such as Bluescript® plasmid derivatives (high-copy-number ColEl-based phagemids, Stratagene Cloning Systems Inc., La Jolla, Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeast, or viral vectors such as the baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.). Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, or other known techniques.

[0504] Eukaryotic and prokaryotic host cells, including mammalian cells, suitable for expression of the recombinant Affimer polypeptides disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia piduperi, and the like. pijperi, Pichia stiptis, Pichia methanolica, Pichia sp.), Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa, Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lactunowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa.

[0505] A variety of host-expression vector systems can be utilized to express the recombinant Affimer polypeptides of the present invention. Such host-expression systems represent vehicles in which the coding sequence of a recombinant Affimer polypeptide can be produced and subsequently purified, but also represent cells capable of expressing the Affimer polypeptides of the present invention in situ when transformed or transfected with the appropriate nucleotide coding sequence. These include microorganisms, such as bacteria (e.g., Escherichia coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the Affimer polypeptide coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the Affimer polypeptide coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the Affimer polypeptide coding sequence; and recombinant viral expression vectors (e.g., cauliflower mosaic virus (CμMV) and tobacco mosaic virus (TMV)) containing the Affimer polypeptide coding sequence. These include, but are not limited to, plant cell lines infected with a Ti plasmid or transformed with a Ti plasmid; or mammalian cell lines (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphoid cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (rat retinal cells developed by Crucell)) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0506] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use for the recombinant Affimer polypeptide to be expressed. For example, when large quantities of such protein are to be produced for the generation of a recombinant Affimer polypeptide pharmaceutical composition, vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983) "Easy Identification of cDNA Clones," EMBO J. 2:1791-1794), in which the Affimer polypeptide coding sequence can be individually ligated into the vector in frame with the lacZ coding region to produce a fusion protein; pIN vectors (Inouye et al. (1985) "Up-Promoter Mutations in the Lpp Gene of Escherichia coli," Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989) "Expression of Human Asparagine Synthetase in Escherichia coli," J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express foreign peptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble, and they can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0507] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. This virus is grown in Spodoptera frugiperda cells. The Affimer polypeptide coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0508] Several viral-based expression systems are available for mammalian host cells. When adenovirus is used as an expression vector, the desired Affimer polypeptide coding sequence can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable in infected hosts and capable of expressing immunoglobulin molecules. (See, e.g., Logan et al. (1984) "Adenovirus Tripartite Leader Sequence Enhances Translation of mRNAs Late After Infection," Proc. Natl. Acad. Sci. (USA) 81:3655-3659.) Specific initiation signals may also be required for efficient translation of the inserted Affimer polypeptide coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bitter et al. (1987) "Expression And Secretion Vectors For Yeast," Methods in Enzymol. 153:516-544).

[0509] In addition, a host cell line can be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20, and T47D, CRL7030, and Hs578Bst.

[0510] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered to stably express the antibodies of the present invention. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. Advantageously, this method can be used to engineer cell lines that express the recombinant Affimer polypeptides of the present invention. Such engineered cell lines may be particularly useful for screening and evaluating compounds that interact directly or indirectly with the recombinant Affimer polypeptides.

[0511] Several selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al. (1977) "Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells," Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962) "Genetics of Human Cess Line. IV. DNA-Mediated Heritable Transformation of a Biochemical Trait," Proc. Natl. Acad. Sci. (USA) 48:2026-2034), and adenine phosphoribosyltransferase (Lowy et al. (1980) "Isolation of Transforming DNA: Cloning the Hamster Aprt Gene," Cell 22:817-823) genes, which can be utilized in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance can also be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) "Transformation of Mammalian Cells With an Amplfiable Dominant-Acting Gene," Proc. Natl. Acad. Sci. (USA) 77:3567-3570; O'Hare et al. (1981) "Transformation of Mouse Fibroblasts to Methotrexate Resistance by a Recombinant Plasmid Expressing a Prokaryotic Dihydrofolate Reductase," Proc. Natl. Acad. Sci. (USA) 78:1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al.(1981) "Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase," Proc. Natl. Acad. Sci. (USA) 78:2072-2076; neo, which confers resistance to the aminoglycoside G-418 (Tachibana et al. (1991) "Altered Reactivity of Immunoglobutin Produced By Human-Human Hybridoma Cells Transfected By pSV2-Neo Gene," Cytotechnology 6(3):219-226; Tolstoshev (1993) "Gene Therapy, Concepts, Current Trials And Future Directions," Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) "The Basic Science Of Gene Therapy," Science 260:926-932; and Morgan et al. (1993) "Human gene therapy," Ann. Rev. Biochem. 62:191-217. Methods generally known in the art for recombinant DNA technology that can be used are described in Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY, and Chapters 12 and 13, Dracopoli et al. (eds), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY; Colbere-Garapin et al.(1981) "A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells," J. Mol. Biol. 150:1-14; and hygro, which confers resistance to hygromycin (Santerre et al. (1984) "Expression of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells," Gene 30:147-156).

[0512] The expression level of a recombinant Affimer polypeptide can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells," in DNA CLONING, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the recombinant Affimer polypeptide is amplifiable, increasing the level of inhibitor present in the host cell culture medium will increase the copy number of the marker gene. Because the amplified region is linked to the nucleotide sequence of the recombinant Affimer polypeptide, production of the recombinant Affimer polypeptide will also increase (Crouse et al. (1983) "Expression and Amplification of Engineered Mouse Dihydrofolate Reductase Minigenes," Mol. Cell. Biol. 3:257-266).

[0513] When the Affimer polypeptide is an Affimer antibody fusion or other multiprotein complex, two expression vectors, one or both of which contain the Affimer polypeptide coding sequence, can be cotransfected into host cells: for example, one vector encoding the heavy chain and a second vector encoding a light chain-derived polypeptide. The two vectors can contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. In such situations, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot (1986) "Expression and Amplification of Engineered Mouse Dihydrofolate Reductase Minigenes," Nature 322:562-565; Kohler (1980) "Immunoglobulin Chain Loss in Hybridoma Lines," Proc. Natl. Acad. Sci. (USA) 77:2197-2199). The coding sequences for the heavy and light chains may comprise cDNA or may comprise genomic DNA.

[0514] Generally, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is unique to the glycoprotein produced in that cell line or transgenic animal. Thus, the specific glycosylation pattern of a recombinant Affimer polypeptide will depend on the particular cell line or transgenic animal used to produce the protein. In certain embodiments of Affimer / antibody fusions, a glycosylation pattern that includes only non-fucosylated N-glycans may be advantageous, as antibodies have been shown to typically exhibit stronger efficacy than their fucosylated counterparts both in vitro and in vivo (see, e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775).

[0515] Furthermore, expression of Affimer polypeptides from production cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed, in whole or in part, in European Patent Nos. 0216846, 0256055, and 0323997, as well as in European Patent Application No. 89303964.4. Thus, in an embodiment of the present invention, a mammalian host cell (e.g., CHO) lacks a glutamine synthetase gene, but is grown in the absence of glutamine in the medium when a polynucleotide encoding an immunoglobulin chain contains a glutamine synthetase gene that complements the lack of the gene in the host cell. Such host cells containing a binding agent or polynucleotide or vector as discussed herein, and expression methods as discussed herein for producing a binding agent using such host cells, are part of the present invention.

[0516] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing properly folded, biologically functional proteins. Baculovirus systems for the production of heterologous proteins in insect cells are well known to those skilled in the art.

[0517] Recombinant Affimer polypeptides produced by transformed hosts can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags, such as hexahistidine, maltose-binding domain, influenza coat sequence, and glutathione-S-transferase, can be attached to the protein to allow for easy purification by passage through an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and X-ray crystallography.

[0518] In some embodiments, recombinant Affimer polypeptides produced in bacterial culture can be isolated, for example, by initial extraction from a cell pellet, followed by one or more concentration, salting-out, aqueous ion exchange, or size-exclusion chromatography steps. HPLC can be used for final purification steps. Microbial cells used for expression of recombinant proteins can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0519] IV. Encoded Affimers for In Vitro Delivery An alternative approach to delivering therapeutic Affimer polypeptides is to rely on the body's own production of the therapeutic polypeptide. Numerous clinical studies have demonstrated the utility of in vivo gene transfer into cells using a variety of different delivery systems. In vivo gene transfer explores administering to patients an "encoded Affimer" nucleotide sequence, rather than an Affimer polypeptide. This allows the patient's body to produce the desired therapeutic Affimer polypeptide over an extended period of time and secrete it systemically or locally, depending on the site of production. Gene-based encoded Affimers may offer a highly labor-efficient and cost-effective alternative to the traditional production, purification, and administration of polypeptide versions of Affimer polypeptides. Several antibody expression platforms that can accommodate the delivery of encoded Affimers have been pursued in vivo: these include viral vectors, naked DNA, and RNA. Encoded Affimer gene transfer not only can enable cost savings by reducing the cost of goods and production costs, but it may also potentially reduce the frequency of drug administration. Overall, sustained in vivo production of therapeutic Affimer polypeptides through expression of the encoded Affimer can contribute to (i) broader therapeutic or prophylactic application of Affimer polypeptides in cost-sensitive settings, (ii) improved accessibility of treatment in both developed and developing countries, and (iii) more effective and affordable treatment modalities. In addition to in vivo gene transfer, cells can be harvested from the host (or donor), engineered to produce the Affimer polypeptide using the encoded Affimer sequence, and readministered to the patient.

[0520] Intramuscular antibody gene administration is the most widely evaluated (reviewed in Deal et al. (2015) "Engineering humoral immunity as prophylaxis or therapy" Curr Opin Immunol. 35:113-22) and, when applied to encoded Affimers, also offers the highest clinical translatability and application. Indeed, the unique anatomical, cytological, and physiological properties of skeletal muscle make it a stable environment for long-term encoded Affimer expression and systemic circulation. Skeletal muscle is easily accessible, allowing for multiple or repeated administrations. The abundant blood vascular supply provides an efficient transport system for secreted therapeutic Affimer polypeptides into the circulation. The syncytial nature of muscle fibers allows nucleotides to diffuse from a limited entry site to multiple adjacent nuclei within the fiber. Skeletal muscle fibers are also terminally differentiated cells, and nuclei within the fiber are postmitotic. Therefore, integration into the host genome is not a prerequisite for achieving sustained mAb expression. The liver is another site frequently used for preclinical antibody gene transfer, typically transfected by iv injection, and may also be the site of gene transfer of the encoded Affimer for either local delivery of the Affimer polypeptide (e.g., for the treatment of liver cancer and / or dysplasia) or for production of an Affimer polypeptide that is secreted into the blood vessels for systemic circulation. This organ has a variety of physiological functions, including the synthesis of plasma proteins. This organ may be particularly well suited for in vivo expression of the encoded Affimer.

[0521] Tumors offer another site for targeted transfer of encoded Affimers, either by iv or direct injection / electroporation. Indeed, expression of encoded Affimers within tumors allows for local production of therapeutic Affimer polypeptides, thus obviating the need for high systemic Affimer polypeptide levels that might otherwise be required to penetrate and affect solid tumors. Similar principles apply to the brain, which is often targeted in the context of antibody gene transfer to circumvent the difficulties associated with blood-brain barrier transport, and which is also a target for delivery of encoded Affimers. See, for example, Beckman et al. (2015) "Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors" Cancer 109(2):170-9; Dronca et al. (2015) "Immunomodulatory antibody therapy of cancer: the closer, the better" Clin Cancer Res. 21(5):944-6; and Neves et al. (2016) "Antibody approaches to treat brain diseases" Trends Biotechnol. 34(1):36-48.

[0522] The success of gene therapy has been greatly promoted by the improvement of non-viral and viral gene transfer vectors. A range of physical and chemical non-viral methods have been used to transfer DNA and mRNA into mammalian cells, and a significant number of these have been developed as clinical-stage techniques for both ex vivo and in vivo gene therapy, and are easily adapted to the delivery of the encoded Affimers of the present invention. Briefly, cationic liposome technology can be used, which is based on the ability of amphipathic lipids with positively charged head groups and hydrophobic tails to bind negatively charged DNA or RNA, forming particles that generally enter cells by endocytosis. Some cationic liposomes also contain neutral co-lipids, which are thought to enhance liposome uptake by mammalian cells. For example, Felgner et al. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. MNAS 84:7413-7417; San et al. (1983) “Safety and short term toxicity of a novel cationic lipid formulation for human gene therapy” Hum. Gene Ther. 4:781-788; Xu et al. (1996) “Mechanism of DNA release from cationic liposome / DNA complexes used in cell transfection" Biochemistry 35,:5616-5623; and Legendre et al. (1992) "Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes" Pharm. Res. 9, 1235-1242.

[0523] Similarly, other polycations, such as poly-l-lysine and polyethyleneimine, can be used to deliver encoded Affimers. These polycations complex with nucleic acids through charge interactions and help condense DNA or RNA into nanoparticles, which then become substrates for endosomal-mediated uptake. Several of these cationic nucleic acid complexing technologies are being developed as potential clinical products, including complexes with plasmid DNA, oligodeoxyribonucleotides, and various forms of synthetic RNA. Modified (and unmodified or "naked") DNA and RNA have also been shown to mediate successful gene transfer in some situations and can also be used as systems for delivering encoded Affimers. These include the use of plasmid DNA via direct intramuscular injection and intratumoral injection of plasmid DNA.For example, see Rodrigo et al. (2012) “De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells” PNAS 109:15271-15276; Oishi et al. (2005) “Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages” Chembiochem. 6:718-725; Bhatt et al. (2015) “Microbeads mediated oral plasmid DNA delivery using polymethacrylate vectors: an effectual groundwork for colorectal cancer” Drug Deliv. 22:849-861; Ulmer et al. (1994) Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines” Vaccine 12: 1541-1544; and Heinzerling et al. (2005) “Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy” Hum. Gene Ther.16:35-48.

[0524] Viral vectors are currently used as delivery vehicles in the vast majority of preclinical and clinical gene therapy trials and in the first approved directed gene therapy. See Gene Therapy Clinical Trials Worldwide 2017 (http: / / www.abedia.com / wiley / ). Their primary driving force is their exceptional gene delivery efficiency, which reflects natural evolutionary development. Viral vector systems are attractive for gene delivery because viruses have evolved the ability to cross cell membranes upon infection, thereby delivering encoded nucleic acids, such as Affinite, to target cells. Pioneering the adenovirus system, the field of viral vector-mediated antibody gene transfer has made significant progress over the past few decades. The myriad of successfully evaluated administration routes, preclinical models, and disease indications has fully demonstrated the capabilities of antibody gene transfer, allowing those skilled in the art to readily identify and adapt antibody gene transfer systems and techniques for in vivo delivery of encoded Affimer constructs. Muscle has emerged as the administration site of choice for sustained mAb expression and has likewise become a suitable target tissue for sustained Affimer polypeptide expression. In the context of vector-based intratumoral gene transfer of encoded Affimers, oncolytic viruses offer distinct advantages because they can specifically target tumor cells, boost Affimer polypeptide expression, and amplify therapeutic responses, such as to checkpoint inhibitory or costimulatory agonist Affimer polypeptides.

[0525] In vivo gene transfer of the encoded affiner can also be achieved using non-viral vectors, such as expression plasmids. Non-viral vectors are easily produced and do not appear to induce specific immune responses. Muscle tissue is most often used as the target tissue for transfection because it is well visualized, easily accessible, and muscle cells are long-lived cells. Intramuscular injection of naked plasmid DNA results in transfection of a specific percentage of muscle cells. Using this approach, plasmid DNA encoding cytokines and cytokine / IgG1 chimeric proteins has been introduced in vivo and has positively affected the outcome of (autoimmune) diseases.

[0526] In some cases, transfection efficiency is increased by so-called intravascular delivery, in which case gene delivery and expression levels are increased by inducing short-term transient hypertension in the veins. Specialized blood pressure cuffs can promote local uptake by temporarily increasing vascular pressure and can be adapted for use in human patients for this type of gene delivery. See, for example, Zhang et al. (2001) "Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates" Hum. Gene Ther., 12:427-438.

[0527] Increased efficiency can also be achieved by other approaches, for example, where nucleic acid delivery is improved by the use of chemical carriers (cationic polymers or lipids) or physical approaches (gene gun delivery or electroporation). See Tranchant et al. (2004) "Physicochemical optimization of plasmid delivery by cationic lipids" J. Gene Med., 6 (Suppl. 1):S24-S35; and Niidome et al. (2002) "Gene therapy progress and prospects: nonviral vectors" Gene Ther., 9:1647-1652. Electroporation is particularly considered an interesting technique for nonviral gene delivery. Somiari, et al. (2000) "Theory and in vivo application of electroporative gene delivery" Mol. Ther. 2:178-187; and Jaroszeski et al. (1999) "In vivo gene delivery by electroporation" Adv. Drug Delivery Rev., 35:131-137. Using electroporation, a pulsed electric current is applied to a local tissue region to increase cell permeability, resulting in gene transfer across the membrane. Studies have shown that in vivo gene delivery can be at least 10 to 100 times more efficient with electroporation than without it.See, for example, Aihara et al. (1998) "Gene transfer into muscle by electroporation in vivo" Nat. Biotechnol. 16:867-870; Mir, et al. (1999) "High-efficiency gene transfer into skeletal muscle mediated by electric pulses" PNAS 96:4262-4267; Rizzuto, et al. (1999) "Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation" PNAS 96: 6417-6422; and Mathiesen (1999) "Electropermeabilization of skeletal muscle enhances gene transfer in vivo" Gene Ther., 6:508-514.

[0528] The encoded Affimer can be delivered by a wide variety of gene delivery systems commonly used in gene therapy, including viral, non-viral, or physical gene delivery systems (see, e.g., Rosenberg et al., Science, 242:1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989)). Discussions of methods and compositions for use in gene therapy include Eck et al., in Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Ninth Edition, Hardman et al., eds., McGraw-Hill, New York, (1996), Chapter 5, pp. 77-101; Wilson, *Clin.Exp.Immunol.* 107 (Suppl. 1):31-32, 1997; Wivel et al., *Hematology / Oncology Clinics of North America*, *Gene Therapy*, S.L. Eck, ed., 12(3):483-501, 1998; Romano et al., *Stem Cells*, 18:19-39, 2000, and references cited therein. U.S. Patent No. 6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions. Delivery routes include, for example, systemic administration and in situ administration.

[0529] To efficient...

Claims

1. 1. A polypeptide suitable for use as a scaffold protein, comprising an amino acid sequence having at least 90% identity to amino acid residues 1-98 of SEQ ID NO:1, comprising one or more mutations relative to SEQ ID NO: 1, wherein said one or more mutations relative to SEQ ID NO: 1 are N32G; The polypeptide has a Tm higher than the Tm of a polypeptide having the sequence: MIPRGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLAS TNYYIKVRAG DNKYMHLKVF NGPPGQNADR VLTGYQVDKN KDDELTGF.

2. 2. The polypeptide of claim 1, further comprising at least one heterologous peptide insertion, the heterologous peptide being inserted at at least one of the following positions relative to SEQ ID NO:1: a) 47-<foreign peptide>-55 b) 46-<foreign peptide>-54 c) 46-<foreign peptide>-50 d) 48-<foreign peptide>-50 e) 49-<foreign peptide>-51 f) 50-<foreign peptide>-52 g) 66-<foreign peptide>-85 h) 67-<foreign peptide>-84 i) 70-<foreign peptide>-74 j) 72-<foreign peptide>-74 k) 71-<foreign peptide>-73 l) 72-<foreign peptide>-81 m) 73-<foreign peptide>-80 n) 79-<foreign peptide>-81 o) 80-<foreign peptide>-81 p) 82-<foreign peptide>-83 q) 72-<foreign peptide>-77 r) 73-<foreign peptide>-78 s) 74-<foreign peptide>-79 t) 4-<heterologous peptide>-5 The polypeptide comprising:

3. 3. The polypeptide of claim 2, comprising two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (a)-(f) and a second heterologous peptide insertion at any of positions (g)-(s).

4. 3. The polypeptide of claim 2, comprising two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (a)-(f) and a second heterologous peptide insertion at position (t).

5. 3. The polypeptide of claim 2, comprising two heterologous peptide insertions, a first heterologous peptide insertion at any of positions (g)-(s) and a second heterologous peptide insertion at position (t).

6. 3. The polypeptide of claim 2, comprising three heterologous peptide insertions: a first heterologous peptide insertion at any of positions (a)-(f), a second heterologous peptide insertion at any of positions (g)-(s), and a third heterologous peptide insertion at position (t).

7. The polypeptide of any one of claims 1 to 6, further comprising one or more mutations selected from the group consisting of G4R, G4W, V48D, V48E, G50S, Y35W, Y43W, Y53W, Y54W, Y64W, F70W, Y85W, F98W, (K71N and S72G and L73P), and (E78A and L80R) relative to SEQ ID NO:

1.

8. A polypeptide according to any one of claims 1 to 7, comprising 5 or fewer mutations relative to SEQ ID NO:

1.

9. 9. The polypeptide of claim 8, wherein the five or fewer mutations are selected from the group consisting of N32G, Y35W, V48D, M65I, Q42E, and T51L.

10. 9. The polypeptide of claim 8, wherein the five or fewer mutations are selected from the group consisting of N32G, V48D, M65I, Q42E, and T51L.

11. i) N32G and V48D; or ii) N32G, V48D, and M65I; or iii) N32G, V48D, M65I, and T51L; or iv) N32G, V48D, M65I, and Q42E; or v) N32G, V48D, M65I, Q42E, and T51L 11. The polypeptide of claim 10, having each of the mutations in one of the groups:

12. 12. The polypeptide of claim 11 having each of the following mutations: N32G, V48D, M65I, and Q42E.

13. a) N32G, Y35W, V48D, M65I, Q42E, T51L, (A59V, G60N, ΔD61, and N62G); and b) N32G, Y35W, V48D, M65I, Q42E, T51L, (A59V, G60N, ΔD61, and N62G), (E29K, K30E, and E33K) 8. The polypeptide according to claim 1, having each of the mutations in one of the groups:

14. 14. The polypeptide of claim 13, having each of the following mutations: N32G, Y35W, V48D, M65I, Q42E, T51L, (E29K, K30E, and E33K).

15. The polypeptide of any one of claims 2 to 14, wherein the heterologous peptide is 6 to 36 amino acids in length.

16. a. A polypeptide according to any one of claims 1 to 15; b. A secretion signal sequence, a peptide linker sequence, an affinity tag, a transmembrane domain, a cell surface retention sequence, a substrate recognition sequence for post-translational modification, a multimerization domain for generating a multimeric structure of a protein aggregated by protein-protein interaction, a half-life extending polypeptide moiety, a polypeptide sequence that changes the tissue localization and antigen binding site of an antibody, one or more additional polypeptides according to any one of claims 1 to 15 that bind to the same or different targets, and one or more additional AFFIMERs (registered trademarks) that bind to the same or different targets. one or more additional amino acid sequences selected from the group consisting of the target polypeptide sequences; A fusion protein comprising:

17. 17. The fusion protein of claim 16, comprising one or more half-life extending polypeptide moieties selected from the group consisting of an Fc domain or a portion thereof, an albumin protein or a portion thereof, an albumin binding polypeptide moiety, transferrin or a portion thereof, a transferrin binding polypeptide moiety, fibronectin or a portion thereof, and a fibronectin binding polypeptide moiety.

18. The fusion protein of claim 17 , wherein the Fc domain or a portion thereof retains FcN ​​binding.

19. 18. The fusion protein of claim 17, wherein the Fc domain or a portion thereof is from IgA, IgD, IgE, IgG and IgM, or a subclass (isotype) thereof, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

20. 18. The fusion protein of claim 17, wherein the Fc domain or a portion thereof retains an effector function selected from C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of the B-cell receptor, or a combination thereof.

21. 18. The fusion protein of claim 17, wherein the half-life extending polypeptide portion extends the serum half-life of the protein by at least 5-fold compared to the absence of the protein.

22. A polypeptide according to any one of claims 1 to 15, which is an AFFIMER® polypeptide.

23. The fusion protein of any one of claims 16 to 21, wherein the polypeptide is an AFFIMER® polypeptide.

24. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 15 or 22 or a fusion protein according to any one of claims 16 to 21 or 23.

25. A vector comprising the nucleic acid of claim 24.

26. 25. A library comprising a population of polypeptides according to any one of claims 1 to 15 or 22 or fusion proteins according to any one of claims 16 to 21 or 23, wherein at least two individual polypeptides or fusion proteins within said population comprise different heterologous peptide insertions.

27. 27. A library comprising a population of nucleic acids, said nucleic acids comprising nucleotide sequences encoding a population of polypeptides or fusion proteins according to claim 26.

28. A host cell comprising a polypeptide according to any one of claims 1 to 15 or 22, a fusion protein according to any one of claims 16 to 21 or 23, a nucleic acid according to claim 24, or a library according to claim 26 or 27.

29. A polypeptide according to any one of claims 1 to 15 or 22 or a fusion protein according to any one of claims 16 to 21 or 23 for use in medicine.

30. A polypeptide according to any one of claims 1 to 15 or 22 or a fusion protein according to any one of claims 16 to 21 or 23 for use in the manufacture of a medicament.

31. 1. A method for identifying a peptide capable of binding to a desired structure, comprising: (i) providing a polypeptide according to any one of claims 1 to 15 or 22 or a fusion protein according to any one of claims 16 to 21 or 23, comprising a heterologous peptide insertion; (ii) contacting the polypeptide or fusion protein with the desired structure; (iii) monitoring the association between said polypeptide or fusion protein and said desired structure. Steps to Including, The method, wherein association of the polypeptide or fusion protein with the desired structure identifies the peptide as a candidate peptide capable of binding to the structure.

32. Use of a polypeptide according to any one of claims 1 to 15 or 22 or a fusion protein according to any one of claims 16 to 21 or 23 as a scaffold protein.

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