CN117597353A

CN117597353A - SAC7D variants against factor C3 and medical uses thereof for treating complement mediated disorders

Info

Publication number: CN117597353A
Application number: CN202280035570.7A
Authority: CN
Inventors: M·西尼耶; A·谢弗雷尔; O·基滕
Original assignee: AFFILOGIC
Current assignee: AFFILOGIC
Priority date: 2021-03-18
Filing date: 2022-03-18
Publication date: 2024-02-23

Abstract

The present invention relates to polypeptides comprising variants of Sac7d family proteins that specifically bind complement component 3 (C3) and/or component C3b obtained after hydrolysis of C3 and inhibit the complement cascade.

Description

SAC7D variants against factor C3 and medical uses thereof for treating complement mediated disorders

Background

Disruption of complement balance associated with increased production and/or activation of complement molecules can lead to a deregulation of the immune system, the appearance or exacerbation of autoimmune, inflammatory, degenerative, blood-fluid or ischemic conditions. Thus, it is interesting to identify molecules that can inhibit the complement cascade.

WO 2008068637 describes the possibility to identify variants of Sac7d which can bind to proteins.

Goux et al (Bioconjugate Chemistry, vol.28, 9, 2017, 2361-2371) are a document describing the use of anti-EGFR conjugates as in vivo tumor diagnostic agents.

WO2013152024 describes anti-C3 antibodies.

WO2020234432 relates to the treatment of diseases using anti-C5 antagonists.

Disclosure of Invention

In a first aspect, the present invention relates to a polypeptide comprising a variant of a Sac7d family member that binds to C3 and/or C3b, wherein said variant comprises 4 to 20 mutated residues in the binding interface of the Sac7d family member to its natural ligand, wherein said variant comprises W24Y and R42W mutations, the numbering of which corresponds to the Sac7d (SEQ ID NO: 1) residue. In particular, the variants comprise 5 to 14, or 5 to 13, mutant residues in the binding interface of a Sac7d family member with its natural ligand.

Specifically, the polypeptide further comprises at least one of the K9T, S L and A44Y mutations, the numbering of which corresponds to the Sac7d (SEQ ID NO: 1) residue. In one embodiment, the polypeptide further comprises a K9T mutation. In one embodiment, the polypeptide further comprises an S31L mutation. In one embodiment, the polypeptide further comprises an a44Y mutation. In one embodiment, the polypeptide further comprises K9T and S31L mutations. In one embodiment, the polypeptide further comprises K9T and a44Y mutations. In one embodiment, the polypeptide further comprises S31L and a44Y mutations. Specifically, the polypeptide further comprises K9T, S L and A44Y mutations, the numbering of which corresponds to that of the Sac7d residue shown in SEQ ID NO. 1.

In some embodiments, the polypeptide further comprises at least one mutation selected from the group consisting of D16E, N Q and M57L, the numbering of which corresponds to the Sac7D (SEQ ID NO: 1) residue.

Specifically, the mutant residue in the binding interface of a Sac7D family member with its natural ligand is selected from the group consisting of V2, K3, K5, K7, Y8, K9, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40, A44, S46, E47, K48, D49, A50 and P51 of Sac 7D.

Specifically, members of the Sac7d family are selected from Sac7d from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), sac7e from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), SSo d from sulfolobus solfataricus (Sulfolobus solfataricus), ssh7b from sulfolobus shihei (Sulfolobus shibatae), ssh7a from sulfolobus shihei (Sulfolobus shibatae), DBP7 from sulfolobus head Kou Dai (Sulfolobus tokodaii), sis7a from sulfolobus icebergii (Sulfolobus islandicus), mse7 from Methanococcus diligens (Metallosphaera sedula), mcu7 from Methanococcus copper (Metallosphaera cuprina), aho7a from Alternaria hollandii (Acidianus hospitalis), aho7b from Alternaria hollandii (Acidianus hospitalis), aho7c from Alternaria hollandii (Acidianus hospitalis), and Sto7 from Alternaria head Kou Dai (Sulfolobus tokodaii).

In some embodiments, the polypeptide comprises SEQ ID NO 59, SEQ ID NO 45, SEQ ID NO 22, SEQ ID NO 27, SEQ ID NO 17, SEQ ID NO 64, SEQ ID NO 69, SEQ ID NO 74, SEQ ID NO 79, SEQ ID NO 84, SEQ ID NO 89, SEQ ID NO 94 or SEQ ID NO 99.

In some embodiments, the polypeptide comprises SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 92, SEQ ID NO. 93, or amino acids 1-54 of these sequences.

In some embodiments, the polypeptide comprises a variant of a Sac7d family member that binds to C3 and/or C3 b.

In some embodiments, variants of Sac7d family members that bind to C3 and/or C3b are conjugated to an organic molecule.

In some embodiments, the variant of the Sac7d family member that binds to C3 and/or C3b is coupled to another polypeptide, in particular to another variant of the Sac7d family protein.

The invention also relates to nucleic acid molecules encoding said polypeptides, expression vectors comprising such nucleic acid molecules (and including elements allowing transcription in host cells), and host cells comprising the nucleic acid molecules or expression vectors.

The invention also relates to pharmaceutical compositions comprising a polypeptide of the disclosure, a nucleic acid of the disclosure, an expression vector of the disclosure or a host cell of the disclosure, and a pharmaceutically acceptable carrier.

The invention also relates to methods for producing a polypeptide of the disclosure, comprising

a. Culturing a cell culture in which the cells have been transformed with an expression vector of the present disclosure,

and

b. Recovering the polypeptide.

The invention also relates to polypeptides or nucleic acids of the disclosure for use as a medicament.

The invention also relates to polypeptides, nucleic acids, expression vectors or cells of the disclosure for use in treating complement-mediated disorders.

In particular, complement-mediated disorders are characterized by complement-mediated damage to erythrocytes, particularly paroxysmal sleep hemoglobinuria or atypical hemolytic uremic syndrome.

In some embodiments, the complement-mediated disorder is an autoimmune disease, optionally wherein the disorder is multiple sclerosis.

In some embodiments, the complement-mediated disorder relates to the kidney, optionally wherein the disorder is membranous proliferative glomerulonephritis, lupus nephritis, igA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G), or acute kidney injury.

In some embodiments, the complement-mediated disorder involves the central or peripheral nervous system or neuromuscular junction, optionally wherein the disorder is neuromyelitis optica, gillin-barre syndrome, multifocal motor neuropathy, or myasthenia gravis.

In some embodiments, the complement-mediated disorder relates to the respiratory system, optionally wherein the disorder is characterized by pulmonary fibrosis.

In some embodiments, the complement-mediated disorder relates to the vascular system, optionally wherein the disorder is characterized by vasculitis.

The invention also relates to a method of treating a subject having or at risk of a complement-mediated disorder, the method comprising administering to the subject a composition comprising an effective amount of a polypeptide, nucleic acid, expression vector, host cell, or a pharmaceutical composition of the present disclosure.

Detailed Description

In particular, the invention relates to polypeptides comprising Sac7d proteins or variants of Sac7d family proteins, which specifically bind complement component 3 (C3) and/or component C3b obtained after hydrolysis of C3. Specifically, the variants bind to both C3 and C3b. The variants also preferably inhibit the complement cascade and in particular can compete with compstatin (compstatin). Such variants are useful for treating complement-mediated disorders (e.g., in subjects having or at risk of complement-mediated disorders) and/or for modulating complement. Specifically, in certain embodiments, the variant binds to a C3b fragment of a C3 protein after C3 hydrolysis (when the C3b fragment has been released) or before hydrolysis (when the C3b fragment is still within the C3 protein).

In preferred embodiments, such variants comprise SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48 or SEQ ID NO. 49. These sequences are based on the consensus sequences of the Sac7d family, obtained according to the teachings of WO 2012/150114, which WO 2012/150114 shows the portability of possible mutations from one protein of the Sac7d family to another, which proteins exhibit similar structures and binding sites as well as similar amino acid sequences.

In particular, in certain embodiments, the variant is a variant of Sac7d protein and comprises SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53 or SEQ ID NO. 54. In certain embodiments, the variant is a variant of an Sso7d protein and comprises SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, or SEQ ID NO. 58.

It should be noted that the sequences indicated above contain methionine at their N-terminus, but that the invention may also be practiced without the methionine listed at the N-terminus of each sequence. Similarly, amino acids at the ends of the protein may be omitted. In particular, the amino acid located after residue L58 of Sac7d (the residue located after L60 of SEQ ID NO:16 or L59 of Sso7 d) may be omitted. Thus, a variant may comprise amino acids 2-58 of SEQ ID NO. 22, 23, 24, 25, 26, 36, 37, 38, 39, 40, 50, 51, 52, 53 or 54, or amino acids 2-59, 2-60, 2-61, 2-62 or 2-63 of SEQ ID NO. 22, 23, 24, 25, 26, 36, 37, 38, 39, 40, 50, 51, 52, 53 or 54. When based on an Sso7d scaffold, the variant may comprise amino acids 2-59 of SEQ ID NO 27, 28, 29, 30, 41, 42, 43, 44, 55, 56, 57, or 58, or amino acids 2-60 of SEQ ID NO 27, 28, 29, 30, 41, 42, 43, 44, 55, 56, 57, or 58.

When based on Sac7d, the polypeptide may comprise any of SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, or SEQ ID NO 88, or amino acids 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63 of any of these sequences.

When based on Aho7c, the polypeptide may comprise any of SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102 or SEQ ID NO 103, or amino acids 1-56 or 1-57 of any of these sequences.

All of these sequences describe specific variants based on proteins of the Sac7d family. Variants of other proteins in the family can be designed using the alignment in FIG. 1, as described below.

The consensus sequence of proteins Sac7d and Aho7c is SEQ ID NO 104. Variants binding to C3 and/or C3b may also comprise SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72 or SEQ ID NO 73 based on this consensus sequence.

In the case where there is a difference between the specification and the sequence listing, the sequences mentioned in the specification are preferred.

As mentioned above, the specification discloses specifically variants of Sac7d (SEQ ID NO: 1), aho7c (SEQ ID NO: 14), sso7d (SEQ ID NO: 2), but the teachings are also applicable to other proteins of the Sac7d family, in particular Sto7 (SEQ ID NO: 15) which is very similar to Sac7d and Aho7 c. These teachings also apply to other OB folding domains as disclosed in WO 2007139397. The invention is also applicable to SH3 domains, which are small protein domains of about 60 amino acid residues, originally described as conserved sequences in the viral aptamer (aptamer) protein v-Crk, and described in the PFAM database as PF 00018. SH3 domains have a characteristic β -barrel sheet consisting of five or six β -strands arranged in two closely packed antiparallel β -sheets. The linker region may comprise a short helix. It is noted that OB fold and SH3 domains have homology, and that knowledge of the sequence and structure of these domains can be taken into account to determine which amino acids in any OB fold or SH3 domain correspond to the amino acids of Sac7d as disclosed below.

In a specific embodiment, the polypeptide comprises a variant of a protein of the Sac7d family that binds to C3 and/or C3 b.

In a specific embodiment, a variant of a protein of the Sac7d family that binds C3 and/or C3b is linked or fused to another protein or polypeptide. In particular, the other protein or polypeptide may be a variant of the same Sac7d family protein, or may be other variants of a Sac7d family protein, binding to C3 and/or C3b, or binding to another target. In this embodiment, other variants of the Sac7d family are preferred when they bind to albumin.

In particular embodiments, the variant is present in the polypeptide and is therefore covalently linked to other proteins or polypeptides of biological interest via an amine linkage.

In one embodiment, the polypeptide is conjugated to an organic molecule exhibiting some functionality, in particular a kinase inhibitor useful as a VEGF inhibitor.

The invention also relates to genetic constructs comprising a DNA sequence encoding the above polypeptide, vectors comprising such genetic constructs and host cells comprising such genetic constructs in their genome.

The present invention also relates to a method for producing a polypeptide disclosed herein, comprising the steps consisting of

a. Culturing a cell culture in which the cells have been transformed by a genetic construct of the present disclosure,

And

b. recovering the polypeptide.

The invention also relates to the polypeptides disclosed herein for use as a medicament.

The invention also relates to polypeptides disclosed herein for use in the treatment of complement-associated or complement-mediated diseases alone or in combination with another adaptive therapy.

The invention also relates to a method of treating a subject in need thereof, comprising administering to the subject a therapeutic amount of a polypeptide disclosed herein, particularly when the subject has a complement-mediated disease.

The invention also relates to compositions comprising a polypeptide disclosed herein and another agent for simultaneous, separate or sequential (dispersed over a period of time) treatment of complement-associated or mediated diseases. Such other agents are selected from agents known for use in treating complement-associated or mediated diseases.

The compositions are particularly useful when the disease is as disclosed below.

The invention also relates to the use of these variants in therapy, diagnosis or purification. The invention also relates to compositions, in particular oral or topical (skin) compositions, containing said polypeptides or variants.

Note that the sequence of Sac7d is:

MVKVKFKYKGEEKEVDTSKIKKVWRVGKMVSFTYDDNGKTGRGAVSEKDAPK ELLDMLARAEREKK(SEQ ID NO:1)。

the sequence of Sso7d is

MATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPK ELLQMLEKQKK(SEQ ID NO:2)。

The variants disclosed herein that bind to C3 and/or C3b contain mutations at positions 7, 8, 9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44 and/or 46 corresponding to the Sac7d sequence. It is noted, however, that threonine at position 33, as well as threonine at position 40, alanine at position 44, valine at position 26, or serine at position 46 can remain in the variant. However, the variants disclosed herein that bind to C3 and/or C3b contain at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12 mutated amino acids (i.e. amino acids that are different from the corresponding amino acids of SEQ ID NO:1, or from the sequence of the protein from which they are derived from the Sac7d family) compared to SEQ ID NO: 1. There are typically at most 20, more preferably at most 18, more preferably at most 16, 15, 14 or 13 mutated amino acids compared to SEQ ID NO. 1 (or compared to the sequence of the protein from which they are derived from the Sac7d family).

In a preferred embodiment, the polypeptide comprises the sequence SEQ ID NO. 45 or amino acids 2-60 of SEQ ID NO. 45.

MXXXVXFXXXGEEKXVXXSKIXXVYRXGKXXXFXYDXXXGKXGWGXVXEKD APKELXXXLXXXXXXXX(SEQ ID NO:45)。

In the context of the present application, unless otherwise indicated, X may be any naturally occurring amino acid, in particular any standard amino acid (i.e. 20 amino acids encoded in the standard genetic code).

In a preferred embodiment, the polypeptide comprises the sequence

MXXXVXFXXXGEEKXVDXSKIXXVYRXGKXXXFXYDXXXGKXGWGXVXEKD APKELXXXLXXXXXXXX (SEQ ID NO: 17), or amino acids 2-60 of SEQ ID NO: 17. Amino acid D17 may be specifically modified to D17E (SEQ ID NO: 31). Amino acids 2-60 of SEQ ID NO. 31 may also be used.

These sequences are based on SEQ ID NO. 16, SEQ ID NO. 16 being a consensus sequence for a Sac7d family protein. SEQ ID NO. 17 and SEQ ID NO. 31 differ from SEQ ID NO. 16 in that the mutations W25Y (which corresponds to the mutation W24Y in Sac7 d) and R44W (which corresponds to the mutation R42W in Sac7 d) are present. Indeed, the inventors have demonstrated that the deletion of these two amino acids alters the binding capacity of the variant to C3 and/or C3 b. SEQ ID NO. 31 differs further from SEQ ID NO. 16 in that the D17E mutation.

It is further shown that the sequences provided herein contain methionine as the first amino acid. However, the methionine may be omitted in the polypeptides disclosed herein. Furthermore, the C-terminus of the proteins disclosed herein is not necessary to obtain the biological effect of the polypeptide and may be omitted. This is further indicated below.

In these sequences SEQ ID NO. 45, SEQ ID NO. 17 and SEQ ID NO. 31, the amino acids denoted X at positions 8-10, 22-23, 27, 30, 32, 34, 42, 46 and 48 are shown in Table 2. Other amino acids denoted as X are shown in Table 1 or Table 10 (where "-" means no amino acid).

TABLE 1 description of the amino acids in SEQ ID NO 17-SEQ ID NO 21 and SEQ ID NO 31-SEQ ID NO 35 and SEQ ID NO 45-SEQ ID NO 49.

Table 2. Description of the amino acids in SEQ ID NO. 17 and SEQ ID NO. 31 and SEQ ID NO. 45.

In another embodiment, the polypeptide comprises the sequence SEQ ID NO. 46 or amino acids 2-60 of SEQ ID NO. 46.

MXXXVXFXXTGEEKXVXXSKIXXVYRXGKXXLFXYDXXXGKXGWGYVXEKDAPKELXXXLXXXXXXXX(SEQ ID NO:46)。

In another embodiment, the polypeptide comprises the sequence

MXXXVXFXFXGEEKXVDXSKIXXVYRXGKLXYDXXXGGXGYVXXXYVXEKDAPKUXXXXXXXXXX (SEQ ID NO: 18) or amino acids 2-60 of SEQ ID NO:18

Or:

MXXXVXFXFXGEEKXVEXSKIXXVYRXGKLXYDXXXGGGWGYVXKARAGAPKELXXXXXXXXX (SEQ ID NO: 32) or amino acids 2-60 of SEQ ID NO: 32.

The properties of amino acid X are shown in tables 1, 3 and 10.

Table 3 depicts the amino acids in SEQ ID NO. 18 and SEQ ID NO. 32 and SEQ ID NO. 46.

In a preferred embodiment, the polypeptide comprises the sequence SEQ ID NO. 47 or amino acids 2-60 of SEQ ID NO. 47.

MXXXVXFXATGEEKXVXXSKIXXVYRXGKDXLFSYDXXXGKIGWGYVSEKDAPKELXXXLXXXXXXXX(SEQ ID NO:47)

In another embodiment, the polypeptide comprises the sequence

MXXXVXFXFXGEEKXVDXSKIXXVYRXGKDXLXGKIGGWGYVSEKDAPKELXXXXXXXXXXX (SEQ ID NO: 19) or amino acids 2-60 of SEQ ID NO:19

Or:

amino acids 2-60 of MXXXVXFXFXGEEKXVEXSKIXXVYRXGKLXYDXXXGGWGYVXXYVXEKDAPKLXXXXXXXXXXX (SEQ ID NO: 33) or SEQ ID NO: 33.

The properties of amino acid X are shown in tables 1, 4 and 10.

Table 4. Description of the amino acids in SEQ ID NO. 19 and SEQ ID NO. 33 and SEQ ID NO. 47.

In a preferred embodiment, the polypeptide comprises the sequence SEQ ID NO. 48 or amino acids 2-60 of SEQ ID NO. 48.

MXXXVXFDATGEEKXVXXSKISAVYRTGKDXLFSYDXXXGKIGWGYVSEKDAPKELXXXLXXXXXXXX(SEQ ID NO:48)

In another embodiment, the polypeptide comprises the sequence

MXXXVXVXFDATGEEKXVDXSKISAVYRTGKDXLFSYDXGKIGWGYVSEKDAPKELXXXXXXXXXXXXX (SEQ ID NO: 20) or amino acids 2-60 of SEQ ID NO:20

Or (b)

MXXXVXVXVXVEXSGEEKXVEXSAKVYRTGKDYSYSXGKIGWGYVSEKDAPKELXXXXXXXXXXX (SEQ ID NO: 34) or amino acids 2-60 of SEQ ID NO: 34.

The properties of amino acid X are shown in Table 1.

In a preferred embodiment, the polypeptide comprises the sequence SEQ ID NO. 49 or amino acids 2-60 of SEQ ID NO. 49.

MXXXVXFAATGEEKXVXXSKIANVYRVGKDXLFSYDXXXGKIGWGYVSEKDAPKELXXXLXXXXXXXX(SEQ ID NO:49)

In a further embodiment, the polypeptide comprises the sequence

MXXXVXVXFDATGEEKXVDXSKISAVYRTGKDXLFSYXGKIGWGYVSEKDAPKELXXXXXXXXXXXXX (SEQ ID NO: 21) or amino acids 2-60 of SEQ ID NO:21

Or (b)

MXXXVXFAATGEEKXVDXSKIANVYRVGKDYSYSXGKIGWGYVSEKDAPKELXXXXXXXXX (SEQ ID NO: 35) or amino acids 2-60 of SEQ ID NO: 35.

The properties of amino acid X are shown in Table 1.

Description of amino acids in SEQ ID NO:45 to SEQ ID NO: 49.

Position of	Amino acids
		17	X is E or D

In a further embodiment, the polypeptide comprises the sequence

MVKKKFXXXGEEKEVSKIXXVYRXGXVXFXYDGXGXWGXVXEKDAPKELLDLLARAERE (SEQ ID NO: 50) or amino acids 2-58 of SEQ ID NO: 50.

In a further embodiment, the polypeptide comprises the sequence

MVKKKFXXXGEEKEVSKIXXVYRXGGXVXFXYDGGXWGXVXEKDAPKELLDMLARAERE (SEQ ID NO: 22) or amino acids 2-58 of SEQ ID NO: 22.

Or (b)

MVKKKFXXXGEEKEVETSKIXXVYRXGGXFXFXGGXGXWGXVXEKDAPKELLDMLARAERE (SEQ ID NO: 36) or amino acids 2-58 of SEQ ID NO: 36.

The properties of amino acid X are shown in tables 5 and 11.

Table 5. Description of amino acids in SEQ ID NO. 22 and SEQ ID NO. 36 and SEQ ID NO. 50.

Position of	Amino acids	Preferred amino acids
			7	X may be any amino acid	D. E A, V or P
8	X may be any amino acid	A. L, I, V, T or H
			9	X may be any amino acid	T、I
21	X is A, S, T, V, I, L, Y or F	F. Y, A, L or S
			22	X can be any naturally occurring amino acid	H、N、A、K、Q
26	X is S, T, V, I, L, Y or F	I. L, Y, V or T
			29	X may be any amino acid	D. E, S or A
31	X is L, I, V, Y, F, M or H	L, Y, I or M
			33	X is S, T or A	S, T or A
40	X is T or I	T or I
			44	X is F, Y, I, V, L, M or H	Y、F
46	X is S, T, H or R	S

In a further embodiment, the polypeptide comprises the sequence

MVKKKKFXXTGEEKEVXKIXXVYRXGKXFXYDGXGYXFKAGVEKKEPHELLLDLLARAERE (SEQ ID NO: 51) or amino acids 2-58 of SEQ ID NO: 51.

In a further embodiment, the polypeptide comprises the sequence

MVKKKFXXTGEEKEVDTSKIXXVYRXGKXVLFXGGWGYXEKDAPKELLDMLARAERE (SEQ ID NO: 23) or amino acids 2-58 of SEQ ID NO: 23.

Or (b)

MVKKKKFXXTGEEKEKEVETSKIXXVYRXGKXVLFXGQGGYVXKQKEDAPKELLDMLARAERE (SEQ ID NO: 37) or amino acids 2-58 of SEQ ID NO: 37.

The properties of amino acid X are shown in tables 6 and 11.

Table 6 description of the amino acids in SEQ ID NO. 23 and SEQ ID NO. 37 and SEQ ID NO. 51.

In a further embodiment, the polypeptide comprises the sequence

MVKKKFTGEEKEVXKIXXVYRXGKDXQDGKGWGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 52) or amino acids 2-58 of SEQ ID NO: 52.

In a further embodiment, the polypeptide comprises the sequence

MVKKKFTGEEKEKEVDTSKIXXVYRXGKDVLFSDNGKIGWGYVSEKDAPKELLDMLARAERE (SEQ ID NO: 24) or amino acids 2-58 of SEQ ID NO: 24.

Or (b)

MVKKKFTGEEKEKEVETSKIXXVYRXGKDVLFSYDQGKIGYVSEKDAPKELLDMLARAERE (SEQ ID NO: 38) or amino acids 2-58 of SEQ ID NO: 38.

The properties of amino acid X are shown in tables 7 and 11.

Table 7. Description of amino acids in SEQ ID NO. 24 and SEQ ID NO. 38 and SEQ ID NO. 52.

Position of	Amino acids	Preferred amino acids
			7	X may be any amino acid	D. E A, V or P
21	X is A, S, T, V, I, L, Y or F	F. Y, A, L or S
			22	X can be any naturally occurring amino acid	H、N、A、K、Q
26	X is S, T, V, I, L, Y or F	I. L, Y, V or T

In a further embodiment, the polypeptide comprises the sequence

MVKKKKFDATGEEKEVSKISAVYRTGKDSLQDXGKIGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 53) or amino acids 2-58 of SEQ ID NO:53 (see also Table 11)

In a further embodiment, the polypeptide comprises the sequence

MVKVKFDATGEEKEVDTSKISAVYRTGKDVLFSYDDNGKIGWGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 25) or amino acids 2-58 of SEQ ID NO: 25.

Or (b)

MVKVKFDATGEEKEVETSKISAVYRTGKDVLFSYDDQGKIGWGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 39) or amino acids 2-58 of SEQ ID NO: 39.

In a further embodiment, the polypeptide comprises the sequence

MVKKKVAATGGEEKEVXKEVKIANVYRVGKDGVLFSDXGKIGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 54) or amino acids 2-58 of SEQ ID NO:54 (see also Table 11)

In a further embodiment, the polypeptide comprises the sequence

MVKVKFAATGEEKEVDTSKIANVYRVGKDVLFSYDDNGKIGWGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 26) or amino acids 2-58 of SEQ ID NO: 26.

Or (b)

MVKVKFAATGEEKEVETSKIANVYRVGKDVLFSYDDQGKIGWGYVSEKDAPKELLDLLARAERE (SEQ ID NO: 40) or amino acids 2-58 of SEQ ID NO: 40.

Table 11. Description of amino acids in SEQ ID NO. 50 to SEQ ID NO. 54.

Position of	Amino acids
		16	X is E or D
37	X is Q or N

For all of the above sequences, such polypeptides may be polypeptides further comprising K (lysine) at the C-terminus following the "ERE" mode.

In a further embodiment, the polypeptide comprises the sequence

MATKFXXXGEEKEVXISKIXXVYRXGKXXXYDEGGKGXGXXXVXEKDAPKELLQLLEKQ (SEQ ID NO: 55) or amino acids 2-59 of SEQ ID NO:55 (see also Table 12).

In a further embodiment, the polypeptide comprises the sequence

MATKFXXXGEEKEVDISkiXXVYRXGGKXXXYDEGGKGXGXWGXXVXEFKEDAPKELLQLLEKQ (SEQ ID NO: 27) or amino acids 2-59 of SEQ ID NO: 27.

Or (b)

MATKFXXXGEEKEVEISKIXXVYRXGKXXXYDEGGKGXGXXVXXXFKEDAPKELLQLLEKQ (SEQ ID NO: 41) or amino acids 2-59 of SEQ ID NO: 41.

The properties of amino acid X are shown in Table 8.

Table 8 description of the amino acids in SEQ ID NO. 27 and SEQ ID NO. 41 and SEQ ID NO. 55.

Position of	Amino acids	Preferred amino acids
			7	X may be any amino acid	D. E A, V or P
8	X may be any amino acid	A. L, I, V, T or H
			9	X may be any amino acid	T、I
21	X is A, S, T, V, I, L, Y or F	F. Y, A, L or S
			22	X can be any naturally occurring amino acid	H、N、A、K、Q
26	X is S, T, V, I, L, Y or F	I. L, Y, V or T
			29	X may be any amino acid	D. E, S or A
31	X is L, I, V, Y, F, M or H	L, Y, I or M
			33	X is S, T or A	S, T or A
41	X is T or I	T or I
			45	X is F, Y, I, V, L, M or H	Y、F
47	X is S, T, H or R	S

In a further embodiment, the polypeptide comprises the sequence

MATKFTGEEKEVXISKIXXVYRXGKDILFSYGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 56) or amino acids 2-59 of SEQ ID NO:56 (see also Table 12).

In a further embodiment, the polypeptide comprises the sequence

MATKFTGEEKEKEKEVDISKIXXVYRXGKDILFSEGGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 28) or amino acids 2-59 of SEQ ID NO: 28.

Or (b)

MATKFTGEEKEKEVEISKIXXVYRXGKDILFSYGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 42) or amino acids 2-59 of SEQ ID NO: 42.

The properties of amino acid X are shown in Table 9.

Table 9 depicts the amino acids in SEQ ID NO. 28 and SEQ ID NO. 42 and SEQ ID NO. 56.

In a further embodiment, the polypeptide comprises the sequence

MATKFDATGEEKEVXISKISISAVERTGKDILFSYDGGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 57) or amino acids 2-59 of SEQ ID NO:57 (see also Table 12).

In a further embodiment, the polypeptide comprises the sequence

MATVKFDATGEEKEVDISKISAVYRTGKDILFSYDEGGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 29) or amino acids 2-59 of SEQ ID NO: 29.

Or (b)

MATVKFDATGEEKEVEISKISAVYRTGKDILFSYDEGGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 43) or amino acids 2-59 of SEQ ID NO: 43.

In a further embodiment, the polypeptide comprises the sequence

MATKFAATGGEEKEVXISKIANVYRVGKDILFSEGGGKIGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 58) or amino acids 2-59 of SEQ ID NO:58 (see also Table 12).

In a further embodiment, the polypeptide comprises the sequence

MATVKFAATGEEKEVDISKIANVYRVGKDILFSYDEGGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 30) or amino acids 2-59 of SEQ ID NO: 30.

Or (b)

MATVKFAATGEEKEVEISKIANVYRVGKDILFSYDEGGGKIGWGYVSEKDAPKELLQLLEKQ (SEQ ID NO: 44) or amino acids 2-59 of SEQ ID NO: 44.

Table 12. Description of amino acids in SEQ ID NO:55 to SEQ ID NO: 58.

Position of	Amino acids
		16	X is E or D

When based on the Sac7d scaffold, the preferred sequences are SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 39 or SEQ ID NO. 40, or amino acids 2-58 of these sequences.

The most preferred sequences are SEQ ID NO 39 or SEQ ID NO 40, or amino acids 2-58 of these sequences.

When based on an Sso7d scaffold, preferred sequences are SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 43 or SEQ ID NO. 44, or amino acids 2-59 of these sequences.

The variants disclosed herein that bind to C3 and/or C3b contain 4 to 20 mutations compared to the wild-type corresponding protein. These mutations are preferably located at positions corresponding to positions 7, 8, 9, 21, 22, 26, 29, 31, 33, 40, 44 and 46 of the Sac7 sequence. In the mutation, it is notable that the variant contains tyrosine (Y) at the position corresponding to position 24 of Sac7d and tryptophan (W) at the position corresponding to position 42 of Sac7 d. It has indeed been demonstrated that the deletion of these two amino acids alters the C3/C3b binding.

In some embodiments, the variant further comprises glutamic acid (E) at a position corresponding to 16 of Sac7d and/or glutamine (Q) at a position corresponding to 37 of Sac7 d. These mutations are not necessary for binding to C3/C3b, but improve the pharmacological properties of the variants.

As seen in the above sequence, valine 26 and/or serine 46, and threonine 33 and/or threonine 40 and/or alanine 44 can be retained when the polypeptide is based on the Sac7d scaffold.

Sac7d protein family

The Sac7d family is defined as being related to the Sac7d protein and corresponds to the 7kDa family of DNA binding proteins isolated from extreme bacteria. It is disclosed herein as a representative species of OB fold domain, which is preferably used in the context of the present invention. Since the SH3 domain has homology to the OB folding domain, the teachings related to Sac7d also apply to SH3 scaffolds.

These proteins and this family are described in particular in WO 2008/068637. Thus, in the context of the present invention, a protein belongs to the Sac7d family when it has one of the sequences SEQ ID NO. 1 to SEQ ID NO. 15, or when it has a sequence corresponding to the sequence SEQ ID NO. 16, said sequence SEQ ID NO. 16 being a consensus sequence (obtained from SEQ ID NO. 1 to SEQ ID NO. 9 and SEQ ID NO. 12 to SEQ ID NO. 15). In this consensus sequence, dashes-indicate no amino acids and proteins are not all of the same size). Such Sac7d families include, inter alia, the Sac7d or Sac7e protein from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), the Sso7d protein from sulfolobus solfataricus (Sulfolobus solfataricus), the DBP7 protein (also known as Sto7 protein) from sulfolobus cephali (Sulfolobus tokodaii), the Ssh7b protein from sulfolobus shibatae (Sulfolobus shibatae), the Ssh7a protein from sulfolobus shibatae (Sulfolobus shibatae), the Mse7 from Methaerococcus diligens (Metallosphaera sedula), the Mcu7 from Methaococcus acidi, the Aho7a or Aho7b or Aho7c from Alternaria betica (Acidianus hospitalis), the Sis7a or Sis7b from sulfolobus iceptis (Sulfolobus islandicus), and the p7ss protein from sulfolobus solfataricus (Sulfolobus solfataricus). In view of the wide similarity in the sequences of Sac7d family proteins, the amino acids of another protein, other than Sac7d, corresponding to a given amino acid of Sac7d can be directly and easily identified. In particular, the teaching of WO 2008/068637 can also be used, which shows (in the figures) and explains (in the description) that such proteins are stackable. The content of this document is incorporated herein by reference, in particular with respect to a description of a method for aligning and superimposing various OB fold proteins. As mentioned above, it is useful to use the numbering of amino acids in the Sac7d protein to designate a particular amino acid, and equivalent amino acids can be obtained from FIG. 1.

WO 2012/150114 shows that mutations from one protein of the Sac7d family can be taken into another protein of the same family. This portability (portability) explains the generation of mutants of one protein of the Sac7d family starting from mutants of another protein of the Sac7d family. The first mutant may in particular be obtained by carrying out the method of WO 2008/068637. Thus, it is possible to obtain mutants of any protein of the Sac7d family starting from mutants of any other protein of this family, in particular using the teachings of WO 2012/150114 and fig. 1. Illustratively, for the mutant of Sac7d, the mutant amino acids of the Sac7d mutant may be introduced into the scaffold of another protein using the sequence alignment of FIG. 1. As an example, sso7d variants obtained from Sac7d variants disclosed herein are described as SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, and SEQ ID NO 58.

Using the consensus sequence, variants are described by SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48 or SEQ ID NO. 49.

The number of mutated residues introduced in the wild-type protein sequence in order to obtain the variant is preferably between 4 and 25, or more particularly between 4 and 22 or between 4 and 20. Thus, variants may be obtained which preferably have at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7 or 8, even more preferably at least 10, but generally less than 25, more preferably less than 22, even more preferably less than 20 or less than 15 or 14 substituted amino acids compared to the wild-type OB fold protein (or domain). It should be noted that all and any ranges (e.g., 5-20 or 7-25, etc.) are contemplated herein. Particularly preferred ranges are 4-20, 4-17 and 6-17, 4-14 and 6-14.

Preferably, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids are mutated at the binding site of the OB folding domain relative to the wild-type OB folding domain. Thus, these mutations are preferably introduced at the amino acids corresponding to V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50 and P51 of Sac7D (SEQ ID NO: 1).

In particular, it is of interest when the number of mutated amino acids is between 7 and 14 (inclusive). In particular, the variant may further comprise an amino acid insertion as described in WO 2008/068637.

As shown, the Sac7d family proteins are Sac7d or Sac7e from sulfolobus acidophilus (Sulfolobus acidocaldarius), sso7d from sulfolobus solfataricus (Sulfolobus solfataricus), DBP7 (also known as Sto 7) from sulfolobus first Kou Dai (Sulfolobus tokodaii), ssh7b from sulfolobus shii (Sulfolobus shibatae), ssh7a from sulfolobus shii (Sulfolobus shibatae), mse7 from Metallophycoccus helophoroides (Metallosphaera sedula), mcu7 from Metallophycocus acidophilus (Metallosphaera cuprina), aho7a or Aho7b or Aho7c from Alternaria acidophilus (Acidianus hospitalis), sis7a or Sis7b from sulfolobus iceptis (Sulfolobus islandicus), and p7ss from sulfolobus solfataricus (Sulfolobus solfataricus). The respective sequences of Sac7d, sso7d, sac7e, ssh7b, ssh7a, DBP7, sis7a (3 alleles), mse7, mcu, aho7a, aho7b, aho7c and Sto7 proteins are represented by SEQ ID NO:1 to SEQ ID NO:15, respectively.

The variant of the Sac7d family protein may be referred to as Nanofitin. Thus, the invention is preferably practiced with variants of the proteins represented by SEQ ID NO. 1 to SEQ ID NO. 15, or with the sequence read as SEQ ID NO. 16 (consensus sequence), in particular variants of Sac7 d.

Ligation of Sac7d protein with OB folding protein and SH3 Domain

OB fold proteins are known in the art. They are described in particular in the documents cited above and in Arcus (Curr Opin Struct biol.2002, month 12; 12 (6): 794-801). The OB is folded in the form of a cylinder with five beta (β) sheets. Most OB fold proteins use the same binding interface for their natural ligands, which may be oligosaccharides, oligonucleotides, proteins, metal ions or catalytic substrates. The binding interface mainly comprises residues located in the β -sheet. Certain residues located in the loop may also be involved in binding of the OB fold protein to its natural ligand. Thus, applications WO2007/139397 and WO2008/068637 (2002, supra) describe OB fold protein domains for binding to their natural ligands.

In particular, the document WO2008/068637 describes exactly how to identify the binding domain of an OB-fold protein. Using WU-Blast2 (Lopez et al, 2003,Nucleic Acids Res 31, 3795-3798), T-COFFEE (Notlendame et al, 2000,J Mol Biol 302,205-217) and DALIlite (Holm and Park,2000,Bioinformatics 16,566-567), the position of the binding domain, in particular amino acids that can be modified, can be identified by superimposing several sequences of proteins with OB folding domains and 3D structures. The sequence of Sac7D (SEQ ID NO: 1) is taken as reference, which is the residue V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D35, D36, N37, G38, K39, T40, G41, R42, A44, S46, E47, K48, D49, A50 and P51.

WO 2008/068637 describes that stacking of 3D structures of OB fold proteins or domains (10 domains are used in this application, including Sac 7D) can be performed using DALI website (http:// www.ebi.ac.uk/DALI/interactive. Html) (Holm and Sander,1998,Nucleic Acids Res 26,316-319). Thus, for any OB fold protein (or any OB fold domain), the amino acids involved in the binding site and corresponding to the Sac7d amino acids described above are readily identified. Thus, amino acids are provided that can be mutated in one of these proteins so that amino acids corresponding to any other OB folding domain can be identified.

It is also noted that the OB folding domain is similar to the SH3 domain, so that the equivalent of the Sac7d amino acid in the SH3 domain can be identified.

Examples of OB folding or SH3 Domains

Non-limiting examples of OB-fold proteins that can be used according to the invention are Sac7d, sso7d, the N-terminal domain of SEB (Papageorgiou et al, 1998), chain A of Shiga-like toxin IIe (PDB 2 bosa), human neutrophil-activin peptide 2 (NAP-2, PDB 1 tvxA), molybdenum binding protein (modg) of Azotobacter vinelandii (Azotobacter vinelandii) (PDB 1h9 j), the N-terminal domain of SPE-C (Roussel et al, 1997), the B5 subunit of E.coli Shiga-like toxin (Kitov et al, 2000), cdc13 (Mitton-Fry et al, 2002), the cold shock DNA binding domain of human Y-box protein YB-1 (Kloks et al, 2002), E.coli inorganic pyrophosphatase EPPase (Samygina et al, 2001) or (Arcus, 2002) paper, such as 1krs (lysyl-tRNA synthetase LysS, E.coli), 1C0aA (Asp-tRNA synthetase, E.coli), 1B8aA (Asp-tRNA synthetase, P.kodakaraensis), 1lylA (lysyl-tRNA synthetase LysU, E.coli), 1quqA (replication protein A,32kDa subunit, human), 1quqB (replication protein A,14kDa subunit, human), 1jmcA (replication protein A,70kDa subunit (RPA 70) fragment, human), lotc (protein bound to the end of the telomere, O.nova), 3ullA (mitochondrial ssDNA binding protein, human), 1prtF (pertussis toxin S5 subunit, pertussis), 1 pD (pertussis toxin S5 subunit (ATP binding), pertussis), 3chbD (cholera toxin, vibrio cholerae), 1tiiD (heat labile toxin, e.coli), 2bosA (Verotoxin-1/shiga toxin, B-pentamer, e.coli), 1br9 (TIMP-2, human), 1an8 (superantigen SPE-C, streptococcus pyogenes), 3seb (superantigen SPE, staphylococcus aureus (S. Aureus)), 1aw7A (toxic shock syndrome toxin, staphylococcus aureus), 1jmc (major cold shock protein, e.coli), 1bkb (initial translation factor 5a, p.aerohylum), 1sro (S1 RNA binding domain of PNP enzyme, e.coli), 1d7qA (initial translation factor 1, e1F1a, human), 1ah9 (initial translation factor 1, IF1, E.coli), 1B9mA (Mo-dependent transcriptional regulator ModE, E.coli), 1ckmA (RNA guanylate transferase, chlorella virus, PBCV-1), 1a0i (ATP-dependent DNA ligase, phage T7), 1snc (staphylococcal ribozyme, staphylococcus aureus), 1hjp (DNA helicase ruvA subunit, N-terminal domain, E.coli), 1pfsA (gene V protein, pseudomonas phage pf 3), 1gvp (gene V protein, filamentous phage (F1, M13)), 1gpc (gene 32 protein (gp 32) core, phage T4), 1wgjA (inorganic pyrophosphatase, saccharomyces cerevisiae (S.cerevisiae)), and 2prd (inorganic pyrophosphatase), extreme thermophiles (t.thermophilus)).

Non-exhaustive examples of proteins with SH3 domains are signal transduction aptamer proteins, CDC24, CDC25, PI3 kinase, phospholipase, ras gtpase activator protein, vav proto-oncogene, GRB2, p 54S 6 kinase 2 (S6K 2), SH3D21, C10orf76 (possible), star 3, certain myosins, SHANK1, 2, 3, ARHGAP12, C8orf46, tangao 1, integrase, focal adhesion kinase (FAK, PTK 2), proline-rich tyrosine kinase (Pyk 2, cadk, ptk2β) or TRIP10 (cip 4).

Description of variants binding to C3 and/or C3b based on Sac7d and Aho7C

Sac7d and Aho7c are proteins with great similarity. Sto (SEQ ID NO: 15) also has similarities to these proteins.

SEQ ID NO. 104 corresponds to the consensus sequence of amino acids 2-66 of Sac7d (SEQ ID NO. 1) and amino acids 3-60 of Aho7c (SEQ ID NO. 14). The starting amino acids have been omitted because they are not necessary in the protein structure.

XKVKFKYKGEEKEVDXSKIKKVWRVGKMXSFTYDDNGKTGRGAVSEKDAPKE LLXXXXXXXXXX(SEQ ID NO:104)

In this consensus sequence, X (or Xaa) is shown in Table 13.

Table 13. Description of some of the amino acids represented as Xaa in SEQ ID NO: 59-73.

The consensus sequence of variants binding to C3 and/or C3b is expressed as

Table 14: sequences based on variants of the Sac7d-Aho7c consensus sequence. The amino acids represented by X are further described in table 13, table 15 and table 16.

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Table 15 description of amino acids denoted Xaa in SEQ ID NOS: 59-103. It should be noted that this table is used for the sequences described above for Xaa they contain. For some sequences, amino acids are specified at some positions in table 15, so the rows in the table are not applicable. The sequence listing describes Xaa of the second column of the table.

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In these sequences, some amino acids not involved in binding may also be modified in variants without altering the binding and biological properties of the protein. They are shown in Table 16.

Amino acids denoted Xaa in SEQ ID NO: 59-103. Xaa at position 56 is present only in SEQ ID NOS 74-88.

Position of	Amino acids
		15	Xaa is D or E
36	Xaa is N or Q
		56	Xaa is M or L

Description of variants binding to C3 and/or C3b based on Sac7d (SEQ ID NO: 1)

Specific variants based on Sac7d which bind to C3 and/or C3b are described by SEQ ID NO. 74 to SEQ ID NO. 88.

In these sequences, the initial methionine (M) of the Sac7d protein has been omitted. The applicant has indeed demonstrated that the activity of the protein is not altered when the amino acid is deleted. Likewise, the last 7 amino acids can be omitted from the variant and retain activity.

Thus, the proteins described by amino acids 1-57 of any one of SEQ ID NOS.74 to 88 are also variants according to the invention. Proteins described by amino acids 1-58, 1-59, 1-60, 1-61, 1-62, 1-63 of any one of SEQ ID NOS: 74 to 88, which are also variants according to the invention, can also be cited.

Thus, the polypeptide comprises any one of SEQ ID NO:71 to SEQ ID NO:88, or any truncated protein based on these sequences, and as described above.

Such variants are represented as

Table 17: based on the sequence of the variant of Sac7 d. The amino acids represented by X are further described in tables 15 and 16.

As described above, D16 of Sac7D (which is located at position 15 of SEQ ID NO:74 to SEQ ID NO:88, because M1 present in Sac7D has been omitted), N37 of Sac7D (herein at position 36), or M57 of Sac7D (herein at position 56) may be modified, as disclosed in Table 16.

As regards the consensus sequence of Sac7d/Aho7c, any combination of substitutions (numbering is for SEQ ID NO:74 to SEQ ID NO: 88) is foreseen, although the sequence listing does not describe it in its entirety:

as described above, these sequences each contain mutated amino acids Y23 and W41 (corresponding to positions 24 and 42, respectively, of SEQ ID NO:1 containing an N-terminal methionine), unlike the naturally occurring amino acids in Sac7 d.

The applicant has indeed found that these amino acids are present in the various variants bound to C3 and/or C3b, such modifications resulting in reduced binding (affinity loss or binding loss). Other amino acids may be variable under the conditions mentioned in table 15.

Preferred variants contain T8 (corresponding to position 9 of SEQ ID NO: 1), L30 (corresponding to position 31 of SEQ ID NO: 1) and Y43 (corresponding to position 44 of SEQ ID NO: 1).

Very interesting variants are described by SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88. These variants each further contain A7, D28, S32, I39 and S45 (positions 8, 29, 33, 40 and 46, respectively, corresponding to SEQ ID NO: 1).

SEQ ID NO. 87 (designated B10-3) and SEQ ID NO. 82 are of particular interest.

SEQ ID NO. 88 (designated H03-3) and SEQ ID NO. 83 are also of particular interest.

Aho7c (SEQ ID NO: 14) -based protein description

Variants of Aho7C that bind to C3 and/or C3b are described by SEQ ID NO. 89 to SEQ ID NO. 103. As described above, this protein is very similar to Sac7 d. It is also shown and suggested herein that mutations of Sac7d can be taken from Sac7d to another protein (WO 2012/150114).

In these sequences, the initial Methionine and Alanine (MA) of the Aho7c protein (SEQ ID NO: 14) have been omitted. The applicant has indeed demonstrated that the activity of the protein is not altered when these amino acids are deleted. Likewise, the last 4 amino acids can be omitted from the variant and retain activity.

Thus, the proteins described by amino acids 1-55 of any one of SEQ ID NOS: 89 to 103 are also variants according to the invention. Proteins described by amino acids 1-56, 1-57, 1-58 of any of SEQ ID NOS: 89 to 103, which are also variants according to the invention, can also be cited.

Thus, the polypeptide comprises any one of SEQ ID NO 89 to SEQ ID NO 103, or any truncated protein based on these sequences, and as described above.

Such variants are represented as

Table 18: based on the sequence of the variant of Ao7 c. The amino acids represented by X are further described in tables 15 and 16.

As described above, either D17 of Aho7c (which is located at position 15 of SEQ ID NO:89 to SEQ ID NO:103, because M1A2 present in Aho7c has been omitted), or N38 of Aho7c (which is located at position 36 herein) may be modified, as disclosed in Table 16.

As regards the consensus sequence of Sac7d/Aho7c, any combination of substitutions (numbering is for SEQ ID NO:89 to SEQ ID NO: 103) is foreseen, although the sequence listing does not describe it in its entirety:

as described above, these sequences each contain mutated amino acids Y23 and W41 (corresponding to positions 25 and 43 of SEQ ID NO:14 containing an N-terminal methionine and alanine, respectively) which differ from the naturally occurring amino acids in Aho7 c.

Preferred variants contain T8 (position 10 corresponding to SEQ ID NO: 1), L30 (position 32 corresponding to SEQ ID NO: 1) and Y43 (position 45 corresponding to SEQ ID NO: 14).

Variants of great interest are described by SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103. These variants each further contain A7, D28, S32, I39 and S45 (positions 9, 30, 34, 41 and 47, respectively, corresponding to SEQ ID NO: 14).

SEQ ID NO. 102 (based on B10-3) and SEQ ID NO. 97 are of particular interest.

SEQ ID NO. 103 (based on H03-3) and SEQ ID NO. 98 are also of particular interest.

Production of identified variants

The sequence of the identified variants may be cloned into any suitable vector by any molecular genetic method known in the art.

These recombinant DNA constructs comprising a nucleotide sequence encoding a polypeptide comprising a variant as described above are used in combination with a vector, such as a plasmid, phagemid, phage or viral vector.

These recombinant nucleic acid molecules can be produced by the techniques described in Sambrook et al, 1989 (Sambrook J, fritschi EF and Maniatis T (1989) Molecular cloning: a laboratory manual, cold Spring Harbor Laboratory Press, new York). Alternatively, for example, a synthesizer may be used to chemically synthesize the DNA sequence.

The recombinant construct of the present invention comprises an expression vector capable of expressing RNA, thus producing a protein from the above-described genetic sequence. Thus, the vector may further comprise regulatory sequences, including a suitable promoter operably linked to the Open Reading Frame (ORF) of the genetic sequences disclosed herein. The vector may further comprise a selectable marker sequence, such as an antibiotic resistance gene. When bacteria are used as expression hosts, specific initiation signals and bacterial secretion signals may also be required to efficiently translate the coding sequences.

Production of molecules

Cells are transfected or transformed with a vector comprising a sequence encoding a polypeptide comprising a variant of the above disclosure.

The cells are cultured under such conditions that the protein is expressed and advantageously secreted. The culture conditions of the cells are those typically used for recombinant antibody production and are known in the art. Such conditions known in the art may also be optimized by those skilled in the art, if desired. Kunert and Reinhart (Appl Microbiol Biotechnol.2016; 100:3451-3461) reviewed these methods and provided adequate references thereto.

Bacteria, phages (Shukra et al Eur J Microbiol Immunol (Bp) 2014;4 (2): 91-98) or eukaryotic production systems may be used.

More preferably eukaryotic cells should be used to obtain appropriate post-translational modifications, such as glycosylation.

In particular, CHO (Chinese hamster ovary) cells, PER.C6 cells (human cell line, pau et al, vaccine.2001 21;19 (17-19): 2716-21), HEK 293b cells (human embryonic kidney cells 293), NS0 cells (non-secretory murine myeloma-derived cell line) or EB66 cells (duck cell line Valneva, french fries) can be used.

The present disclosure also provides host cells containing at least one DNA construct encoding a polypeptide comprising a variant disclosed herein. The host cell may be any cell from which an expression vector is available. As described above, the host cell may be a higher eukaryotic host cell (e.g., a mammalian cell), a lower eukaryotic host cell (e.g., a yeast cell), or a prokaryotic cell (e.g., a bacterial cell).

The recombinant construct is introduced into the host cell by any method known in the art, such as calcium phosphate transfection, lipofection, DEAE, dextran mediated transfection, electroporation, or phage infection. These vectors may be inserted into the genome of the host cell or may remain as extragenomic vectors (e.g., bacterial artificial chromosomes or yeast artificial chromosomes). When introduced into the genome of a cell, such introduction may be random or targeted (homologous recombination, etc.) using methods known in the art.

Bacterial hosts and expression

Useful expression vectors for bacterial use are constructed by inserting recombinant DNA sequences into operably-reading-phases (reading-phase) with functional promoters along with appropriate translation initiation and termination signals. The vector will contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

Suitable prokaryotic hosts for transformation include E.coli, B.subtilis (Bacillus subtilis), salmonella typhimurium (Salmonella typhimurium) and various species within the genera Pseudomonas, streptomyces (Streptomyces) and Staphylococcus (Staphylococcus).

Eukaryotic hosts and expression

Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. In particular, the above-described cells can be used.

Transformed or transfected cells are cultured according to methods known in the art and the polypeptide is recovered from the intracellular or extracellular fraction (depending on whether the polypeptide is secreted).

Separation of molecules

The recombinant protein produced may be isolated from the intracellular or extracellular fraction and purified by any of a variety of known isolation methods using the physical or chemical properties of the protein.

In particular, methods such as precipitation, ultrafiltration, various types of liquid chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography), dialysis, and combinations thereof may be used.

In general, any method known and used to purify recombinant polypeptides is suitable for purifying the molecules disclosed herein.

If a tag (e.g., a polyhistidine tag) is introduced into the recombinant sequence, the tag can be used to purify the molecule. However, in certain embodiments, it is preferred to use affinity to purify the molecule.

In particular, the following facts may be exploited: the molecules produced herein bind to a specific target and such molecules are isolated using any affinity method (affinity column, FACS, beads).

One particular advantage of the molecules disclosed herein is that: they need not be glycosylated to render them active and can therefore be produced in any type of cell, not necessarily in eukaryotic cells. They are particularly easy to produce in bacterial cells.

Modification of variants

The variants described above having the ability to bind C3 and/or C3b may be modified by any method known in the art.

Preparation of polypeptides comprising variants

A DNA sequence may be prepared that contains two coding sequences in-frame, one for the variants disclosed herein and one for the protein or peptide of interest. Thus, the resulting expressed protein will be a polypeptide comprising both proteins. The vector may be constructed in such a way that the vector comprises a sequence encoding a linker located between two proteins in the expressed polypeptide.

Thus, the invention also encompasses polypeptides comprising variants of a protein of the OB fold protein (preferably the Sac7d family) that binds to C3 and/or C3b, fused or linked (preferably by an amine linkage as described above) to another protein or polypeptide.

In a specific embodiment, the other protein or polypeptide comprises another variant of a protein of the Sac7d family, in particular as disclosed herein.

Of particular interest are:

a polypeptide comprising a variant of an OB fold protein of the Sac7d family that binds to C3 and/or C3b fused to a variant of an OB fold protein of the Sac7d family that binds to albumin or PD-L1. In particular, variants of OB fold proteins of the Sac7d family that bind albumin are selected from SEQ ID NO:117, SEQ ID NO:118 or SEQ ID NO:119, and may be bound N-terminal or C-terminal to variants that bind C3 and/or C3 b. The polypeptide comprising SEQ ID NO. 25 fused to SEQ ID NO. 119, or the polypeptide comprising SEQ ID NO. 26 fused to SEQ ID NO. 119, may be specifically located. Of particular interest are polypeptides comprising SEQ ID NO. 25 fused at its C-terminus to the N-terminus of SEQ ID NO. 119 (possibly with a linker). Of particular interest are polypeptides comprising SEQ ID NO. 26 fused at its C-terminus to the N-terminus of SEQ ID NO. 119 (possibly with a linker). Of particular interest are polypeptides comprising SEQ ID NO. 25 fused at its N-terminus to the C-terminus of SEQ ID NO. 119 (possibly with a linker). Of particular interest are polypeptides comprising SEQ ID NO. 26 fused at its N-terminus to the C-terminus of SEQ ID NO. 119 (possibly with a linker). The same is true when SEQ ID NO:119 is replaced with SEQ ID NO:117 or SEQ ID NO: 118.

In another embodiment, the other protein or polypeptide is an antibody. In this embodiment, the variant of the OB folding domain is fused to at least one heavy or light chain of an immunoglobulin monomer, preferably at the N or C terminus of the light or heavy chain. In another embodiment, the variant may be fused to both the heavy chain or the light chain.

To obtain such compounds, genetic constructs comprising a DNA sequence selected from the group consisting of:

a. sequences encoding the heavy chain of the antibody fused at its 3' end to sequences encoding variants of the OB folding protein (possibly with sequences encoding linkers)

b. Sequences encoding the heavy chain of the antibody fused at its 5' end to sequences encoding variants of the OB folding protein (possibly with sequences encoding linkers)

c. The sequence encoding the light chain of the antibody is fused at its 3' end to a sequence encoding a variant of the OB folding protein (possibly with a sequence encoding a linker)

d. The sequence encoding the light chain of the antibody is fused at its 5' end to a sequence encoding a variant of the OB folding protein (possibly with a sequence encoding a linker).

Fusion can be performed at the N-terminus and/or C-terminus of the antibody chain (heavy and/or light chain). Notably, particularly when using small OB fold domains (about 70 amino acids), such as proteins from the Sac7d family, molecules with antibody structures (two light chains paired with two heavy chains, such dimers paired together) can be obtained with antibody regions and further with binding regions consisting of modified OB fold domains.

In a specific embodiment, the antibody portion of the proteins disclosed herein is an IgG molecule.

In another embodiment, the antibody portion of the proteins disclosed herein is an IgA molecule.

In another embodiment, the antibody portion of the proteins disclosed herein is an IgM molecule.

In another embodiment, the antibody portion of the proteins disclosed herein is an IgD molecule.

In another embodiment, the antibody portion of the proteins disclosed herein is an IgE molecule.

The antibody may be a human antibody, a rodent antibody (e.g. a mouse or rat antibody), a cat antibody, a dog antibody, a chicken antibody, a goat antibody, a camelid antibody (e.g. a camel antibody, a llama antibody, an alpaca antibody or a nanobody), a shark antibody or an antibody from any other species. The antibody may be chimeric or humanized. As suggested by the wikipedia, humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to naturally occurring antibody variants in humans. Chimeric antibodies comprise sequences from different species.

When an antibody is part of a molecule disclosed herein, it is preferably an antibody comprising two identical heavy chains (about 400 to 500 amino acids, typically about 450 amino acids) and two identical light chains. Thus, the antibodies comprise the same Fab variable region. Thus, the antibody is a monospecific antibody in which both parts of the antibody (combination of light and heavy chains) bind to the same epitope.

However, antibodies may exhibit different heavy and/or light chains. In particular, in some embodiments, the antibody is a bispecific antibody. Thus, the term "antibody" encompasses both the above-described "classical antibodies" having the same heavy and light chains, and engineered antibodies having more than one specificity.

In a specific embodiment, an antibody presents one heavy and light chain from one antibody and another heavy and light chain from another antibody.

The antibody can bind to a target selected from the group consisting of:

cell surface receptor: insulin receptor, low density lipoprotein receptor-related protein 1, transferrin receptor, epidermal growth factor receptor variant III, trkA, trkB, trkC, her, her3, her4, PMSA, IGF-1R, GITR, RAGE, CD.

Cell surface proteins: mesothelin, epCam, CD19, CD20, CD38, CD3, TIM-3, CEA, cMet, ICAM1, ICAM3, madCam, a4b7, CD4, CD138.

Angiogenic factors and growth factors: angiogenin 2, HGF, PDGF, EGF, GM-CSF, HB-EGF and TGF.

Immune checkpoint inhibitors or activators: PD-1, PD-L1, CTLA4, CD28, B7-1, B7-2, ICOS, ICOSL, B7-H3, B7-H4, LAG3, KIR, 4-1BB, OX40, CD27, CD40L, TIM3, A2aR.

Circulating protein: tnfα, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, bace1, alpha synuclein, tau, amyloid.

Alternatively, the polypeptide may comprise a variant as disclosed above and a biologically active molecule, such as erythropoietin, interferon or etanercept.

In another embodiment, variants of the OB folding domain (particularly variants of the Sac7d family protein) are conjugated to an organic molecule. This may be accomplished by any method known in the art. In particular, molecules can be chemically linked to proteins. As molecules, antiproliferative agents (cytotoxic and cytostatic agents) may be cited, including cytotoxic compounds (e.g. broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PBK/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, chaperone inhibitors, HDAC inhibitors, PARP inhibitors, wnt/Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors and proteasome inhibitors. Anti-inflammatory molecules may also be used.

Mention may be made in particular of DNA binding or alkylating agents, such as anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and analogues thereof, alkylating agents, such as calicheamicin, actinomycin, mitomycin (mitromamycin), pyrrolobenzodiazepine, etc. Cell cycle progression inhibitors such as CDK inhibitors, rho kinase inhibitors, checkpoint kinase inhibitors, aurora kinase inhibitors, PLK inhibitors and KSP inhibitors may also be cited. Thalidomide and its derivatives lenalidomide and pomalidomide may also be mentioned. For the treatment of inflammatory diseases cyclooxygenase-2 inhibitors, 5-lipoxygenase inhibitors, quercetin and/or resveratrol may also be used as molecules coupled to polypeptides comprising variants.

Interesting and preferred molecules are also disclosed above.

Use of variants

These variants may be particularly useful in therapeutic methods, particularly for treating complement-mediated or complement-associated diseases.

Accordingly, the present invention relates to methods of treating complement-mediated or complement-associated diseases comprising administering to a subject in need thereof a therapeutic amount of a polypeptide of the disclosure or a variant of the OB fold disclosed herein (particularly a variant of a protein of the Sac7d family). The invention also relates to a method for inhibiting the complement cascade in a subject comprising administering to the subject an effective amount of a polypeptide of the disclosure or a variant of the OB fold disclosed herein (particularly a variant of a protein of the Sac7d family).

As used herein, the term "therapeutic amount" or "effective amount" is an amount sufficient to achieve a beneficial or desired result (e.g., a clinical result), the "effective amount" being dependent on the context of its application. An effective amount is an amount that provides therapeutic improvement while minimizing side effects or adverse effects. The therapeutic improvement may be regression of complement-mediated or complement-associated disease, improvement in quality of life of the subject, improvement in efficacy of the combination therapy. An effective amount may also be an amount that results in inhibition of the complement cascade as clinically observed.

In some embodiments, the variant that binds to C3 may be administered in an amount sufficient to achieve a target concentration in the brain of the subject. In some embodiments, the target concentration of the C3-binding variants described herein (having a length of about 60 to about 66 amino acids) is between about 0.2 micrograms (μg/g) to about 0.375 μg/g per gram of brain tissue. In some embodiments, the target concentration is between about 0.375 μg/g to about 0.75 μg/g. In some embodiments, the target concentration is between about 0.75 μg/g to about 2.0 μg/g. In some embodiments, the target concentration is between about 2.0 μg/g and about 5.0 μg/g. In some embodiments, where a variant that binds to C3 is fused to another polypeptide (e.g., another variant or antibody), a target concentration of the agent equivalent on a molar basis to the variant that binds to C3 may be used.

In some embodiments, for example, to achieve a target concentration in the brain, the dose administered to the human subject may be between about 5 mg/day and about 500 mg/day, e.g., about 5-50 mg/day, about 50-150 mg/day, about 150-300 mg/day, or about 300-500 mg/day. In some embodiments, where a variant that binds to C3 is fused to another polypeptide (e.g., another variant or antibody), a dose of the agent equivalent on a molar basis to the variant that binds to C3 may be used.

The variants may be administered by any method in the art.

In particular, variants may be injected. In another embodiment, the variant may be applied topically (on the skin or eye of the patient), as disclosed in WO 2014/173899. In another embodiment, the variants may be administered orally, as disclosed in WO 2016/062874.

Other routes of administration are also contemplated. In some embodiments, the variant may be administered intravenously or subcutaneously. In some embodiments, the variant may be administered intra-ocularly (e.g., intravitreally) to treat an ocular disorder. In some embodiments, the variant may be administered by intrathecal route (e.g., for treating a disorder affecting the central nervous system). In some embodiments, the polypeptide or variant may be fused or conjugated to a moiety (e.g., an antibody, polypeptide, or small molecule) that binds to a blood brain barrier receptor, which polypeptide or variant crosses the blood brain barrier by receptor-mediated transcytosis. Some such parts are called brain shuttles (shuttles). Blood brain barrier receptors include, for example, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), low density lipoprotein receptor-related protein 8 (LRP 8), low density lipoprotein receptor-related protein 1 (LRP 1), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (such as, for example, tucker, ther. Deliv.2:311-27 (2011); pulgar, front. Neurosci.12:1019 (2019)).

In some embodiments, the polypeptide or variant may be delivered to a subject using an expression vector, such as a viral vector (e.g., a vector suitable for gene therapy), a plasmid vector, a phage vector, a cosmid, a phagemid, an artificial chromosome, and the like. In some embodiments, the nucleotide sequence encoding the polypeptide or variant is integrated into a viral vector. Non-limiting examples of viral vectors include: retroviruses (e.g., moloney Murine Leukemia Virus (MMLV), harvey murine sarcoma virus, murine mammary tumor virus, rous sarcoma virus), adenoviruses, adeno-associated viruses, SV 40-type viruses, polyoma viruses, epstein-Barr viruses, papilloma viruses, herpesviruses, vaccinia viruses, and polioviruses.

In some embodiments, the polypeptide or variant (or nucleotide encoding the polypeptide or variant) may be linked (e.g., physically linked) to a delivery agent, such as a nanoparticle (e.g., a lipid nanoparticle), dendrimer, polymer, liposome, or cationic delivery system.

Variants or polypeptides containing variants may also be used in diagnostic methods. In particular, such variants or polypeptides may be linked to any marker known in the art and used in imaging methods.

The invention therefore also relates to a method for detecting the presence of C3 and/or C3b in a sample, or quantifying it, comprising the steps of:

a. exposing the sample to variants of the disclosure that bind to C3 and/or C3b under conditions that permit binding

b. Recovering the variant and/or detecting, or measuring, the amount of C3 and/or C3b bound to the variant.

b) The recovery of (c) may be carried out by various washing or methods commonly used in the art. Detection or quantification may be performed by any method, such as ELISA, chromatography or fluorescence, or other methods in the art.

Complement-associated or mediated diseases

A complement-associated or complement-mediated disease or condition relates to any disease or condition in which an increase in the complement system is observed and which can result in complement-mediated damage to an organ, tissue or cell of a subject.

The polypeptides disclosed herein may be administered to a subject to inhibit complement alone or in combination with one or other products that also inhibit the complement system or target other disease-related targets, or have therapeutic efficacy in treating or ameliorating a disease or condition.

Can be enumerated by

-protection of Red Blood Cells (RBCs), in particular in paroxysmal sleep hemoglobinuria or atypical hemolytic uremic syndrome;

Protection of transplanted organs, tissues and/or cells (often in combination with other immunosuppressive drugs), in particular reduction of ischemia/reperfusion (I/R) injury

Paroxysmal sleep hemoglobinuria (PNH) is a rare disorder characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure, and thrombotic liability. Atypical hemolytic syndrome (aHUS) is a chronic condition characterized by microangiopathy hemolytic anemia, thrombocytopenia, and acute renal failure, caused by inappropriate complement activation, typically due to mutations in the gene encoding complement regulatory proteins. Complement-mediated hemolysis can also occur in a variety of other conditions, including autoimmune hemolytic anemia, such as primary chronic collectin disease, as well as certain reactions to drugs and other foreign substances.

Transplantation (graft) involves the replacement of organs and tissues, and blood transfusion is herein considered to be "graft". Ischemia reperfusion (I/R) injury occurs when an organ is hypoxic during the process of harvesting from a donor to transplantation into a host. The polypeptides disclosed herein have certain utility in preventing I/R injury and graft rejection. They may be used in a transplant medium, during the transport of the transplanted organ from the donor to the host, or they may be administered to the host after transplantation.

Other complement-mediated or related disorders include ocular disorders such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma or uveitis, autoimmune diseases, particularly when mediated at least in part by antibodies to one or more autoantigens. Rheumatoid arthritis, myasthenia gravis, neuromyelitis optica (NMO, dewiki disease), glomerulopathy of certain kidneys may be cited.

Other diseases that may be treated by the polypeptides disclosed herein include intracerebral hemorrhage, neurodegenerative diseases, allergic or infusion reactions, asthma, sinusitis, nasal polyposis, chronic Obstructive Pulmonary Disease (COPD), or idiopathic pulmonary fibrosis. In some embodiments, the disease is alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, dementia with lewy bodies (i.e., dementia with lewy bodies or parkinson's disease dementia), frontotemporal dementia, traumatic brain injury, traumatic spinal cord injury, multiple system atrophy, chronic traumatic encephalopathy, chronic inflammatory demyelinating polyneuropathy, guillain-barre syndrome, multifocal motor neuropathy, creutzfeldt-jakob disease, chronic pain (e.g., neuropathic pain), or leptomeningeal transfer. Other diseases that may be treated by the polypeptides disclosed herein include chronic inflammatory diseases, which may be autoimmune diseases such as psoriasis, atopic dermatitis; systemic scleroderma and scleroderma; inflammatory Bowel Disease (IBD) (e.g., crohn's disease and ulcerative colitis); behcet's disease; dermatomyositis; polymyositis; multiple Sclerosis (MS); dermatitis is treated; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; rheumatoid Arthritis (RA), sjogren's syndrome, vasculitis; inflammatory diseases of the Central Nervous System (CNS), chronic hepatitis; chronic pancreatitis, glomerulonephritis; sarcoidosis; thyroiditis, pathological immune response of tissue/organ transplantation (e.g., transplant rejection); COPD, asthma, bronchiolitis, hypersensitivity pneumonitis, idiopathic Pulmonary Fibrosis (IPF), periodontitis and gingivitis.

Mention may also be made of diseases affecting the musculoskeletal system (such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthropathies, rette's syndrome, gout), or diseases affecting the circulatory system (vasculitis or other diseases associated with vasculitis, such as vascular and/or lymphangitis, polyarteritis nodosa, wegener granulomatosis, giant cell arteritis, churg-strauss syndrome, microscopic polyangiitis, hodgkin-Song Laiyin purpura, polyarteritis, kawasaki disease, hoton's disease or behcet's disease).

In some embodiments, the disorder is cancer.

Drawings

Fig. 1: alignment of Sac7d family proteins.

Fig. 2: binding to C3b was verified in selected variants that bind to C3.

Fig. 3: based on sequence similarity, clusters of C3 and C3b binders are repartitioned.

Fig. 4: competitive assays were performed on C3b binding of Nanofitin from different clusters using compstatin.

Fig. 5: EC50 for C3b binding of various nanofitines in ELISA.

Fig. 6: inhibition of the complement cascade by various nanofitins.

Fig. 7: the figures depict the concentration of C3 inhibitory NF ("C3 NF") in CSF (left panel) and plasma (right panel) after intrathecal administration of C3 NF to cynomolgus monkeys at different dosage levels.

Fig. 8: the figure depicts the distribution in various brain regions of cynomolgus monkeys after intrathecal administration of C3 inhibitory NF ("C3 NF") at prescribed doses. The "target tissue concentration" depicted in fig. 8 represents a concentration of C3NF that may be desired to be achieved in some circumstances and is not meant to be limiting.

Fig. 9: the figures show the level of C3a in brain tissue after intrathecal administration of C3 inhibitory NF ("C3 NF") or carrier to cynomolgus monkeys, demonstrating functional inhibition of C3 cleavage in cognition-related brain regions of cynomolgus monkeys treated with C3 NF.

Fig. 10: the figure shows the level of C3 inhibitory NF ("C3 NF") in the medium of hiPSC-derived brain cells transfected with an expression plasmid encoding C3 NF. P1 and P2 represent different plasmids.

Fig. 11: the figure shows the level of C3 inhibitory NF ("C3 NF") in the medium of ARPE-19 cells transfected with an expression plasmid encoding C3 NF.

Fig. 12: the figure shows brain levels of C3 inhibitory NF that were able to brain shuttle in rat brain after a single intravenous administration.

Fig. 13: clusters of some C3 and C3b conjugates were repartitioned based on the presence of specific mutations.

Detailed Description

Examples

Example 1 identification of variants that bind to C3 and C3b

The conversion of C3 to C3b is accompanied by a significant structural change.

Using immobilized C3b as a target, 190 variants binding to C3 and C3b were screened by ELISA as crude bacterial supernatant, which made it possible to verify a high proportion of positive binders (fig. 2). Subsequent sequencing showed strong sequence diversity with few repeated sequences. Furthermore, the division of Nanofitin into several clusters, based on its homology to the Nanofitin binding site, highlights the enrichment of specific patterns, where hit rates exceeding 50% were found in clusters 1 and 2 (fig. 3). Such variants based on the Sac7d sequence are called nanofitins.

Example 2 identification of Nanofitin competing with Capstatin

Compstatin is a 13 residue synthetic peptide that binds C3 at Macroglobulin (MG) domains 4 and 5. This binding site is part of the MG loop formed by domains MG1-6, which is structurally conserved for C3 and its truncated variants C3b and C3C. Interestingly, this domain is remote from any other known binding site on C3, suggesting that compstatin inhibitory activity is involved in steric-based mechanisms of action; thus blocking complement activation and amplification by limiting substrate C3 contact with the invertase complex.

Candidates targeting epitopes overlapping with those of compstatin were identified by competitive assays by biolayer interferometry using Nanofitin found specific for C3b and representative for each sequence cluster (clusters 1 to 6). The ratio between the binding reaction on C3b and the binding reaction on C3b complexed with compstatin analogs was used as a measure of the competition potential of the different nanofitines.

As a result, the binding reaction of Nanofitin from cluster 1 to cluster 5 was found to be unaffected by the presence of compstatin analogues (ratio-1), indicating that they bind C3b on a different epitope than compstatin.

The Nanofitin in cluster 6 showed a decrease in binding response in the presence of compstatin analogs (ratio > 1), indicating that compstatin blocks their epitopes (fig. 4).

Further characterization included evaluation of dose response by ELISA, and screening for complement cascade inhibitory activity at four concentrations (1, 0.1 and 0.01 μm) in a classical pathway-based wiesab assay.

Three clones from cluster 6 were found to be able to inhibit the complement cascade, the neutralization potential of which was correlated with their EC50 in ELISA. It was also noted that binding to C3b appeared to be insufficient to trigger effective inhibition of the complement cascade, as Nf4 from cluster 1 appeared to be a weak inhibitor, although its EC50 in ELISA was low. Taken together, these data indicate that clones from cluster 6 demonstrate a functional compstatin-like mechanism of action by targeting similar epitopes and providing similar neutralizing activity. This is illustrated in fig. 5 and 6.

A clone designated Nf1 was further studied and characterized. The sequence of this clone falls within the range of SEQ ID NO. 22.

EXAMPLE 3 characterization/improvement of clone Nf1

Important residues involved specific for C3 were identified.

Other residues are further modified to modulate the rate of dissociation.

Mutants were generated by starting to randomly replace all residues in the original Nanofitin library with alanine (except at position 8, since in Nf1 the residue appears to have been alanine).

The binding capacity of these different variants (at 10nM and 100nM, respectively) was then evaluated in ELISA, with EC100 being 10 times that of Nf 1.

As a result, Y24 and W42 were found to be the most important residues that remained bound to C3b with good affinity.

Positions 9, 31 and 44 were also found to be important (to a lesser extent) for maintaining good affinity with C3 b. The location 46 is found to be more tolerant (permissive).

Analysis of some nanofitines showed that it was possible to identify variants that bind to C3/C3b with the two most important Y24 and W42 mutations (and other mutations not indicated), or with other mutations alone or in combination (reported to be of some importance) (fig. 13).

In contrast, in ELISA, other alanine variants retained binding capacity to C3b at the two concentrations studied, with different levels of influence on their binding response depending on the location involved. The results indicate that the main residues of Nf1 contributing to the C3 specificity are located at positions 24 and 42, and that some possible variations are at positions 9, 31 and 44 and also at position 46.

Other residues are quite tolerant of substitution with alanine, indicating that these residues contribute less importantly or secondarily to specificity.

Note that the primary residues are located in the core of the binding site and the secondary residues are located in the periphery.

The binding was optimized by determining residues at positions 7, 21, 22, 26, 29, 33 and 40, which can increase affinity.

The two clones showing the highest ELISA signal (0 d 450nm > 4) were purified and affinity measurements and neutralization potency were further characterized in the weislab assay based on the alternative and classical pathways. As a result of randomization and subsequent off-rate selection, the 2 variants of Nf1 (SEQ ID NO:25 and SEQ ID NO: 26) were found to exhibit slower off-rates and overall higher affinities than Nf1 itself.

The binding kinetics parameters of anti-C3 Nanofitin were measured by interferometry on the Octet RED96 system (ForteBio). Streptavidin biosensors were first functionalized with 2nm biotinylated anti-GFP Nanofitin (10. Mu.g/mL in TBS containing 0.002% Tween 20 and 0.01% BSA) and then loaded with 1.5nm anti-C3 GFP tagged Nanofitin (10. Mu.g/mL in TBS containing 0.002Tween 20 and 0.01% BSA). The biosensor was allowed to equilibrate for 60 seconds and then the binding kinetics were assessed by simultaneously exposing the biosensor to different concentrations (150, 50, 16.6, 5.55 and 1.85 and 0 nM) of TBS solution of C3b (containing 0.002% Tween 20 and 0.01%). The association and dissociation steps were each measured for 180 seconds. Three cycles of 10 seconds of alternate washes with glycine (10 mM, pH 2.5) and TBS were used to regenerate the biosensor. All steps were run at 30℃with a continuous shaking speed of 1000 RPM. Biosensors exposed to 0nM concentration were used as background references. The sensorgrams were processed using single reference subtraction and analyzed using the Octet data analysis software 11.1 (ForteBio). Fitting was performed using a 1:1 binding fitting model.

As demonstrated by the weislab assay, this increase in affinity (table 19) translates to higher neutralization potency compared to Nf 1.

Table 19 affinity measurement of the optimized C3-binders.

Example 4 these variants are tolerant to fusion at both the N-and C-termini

The paratope and the N-terminal and C-terminal endpoints of Nanofitin are located on opposite sides. Thus, nanofitin can participate in fusion constructs without altering binding to its target, which can include gene fusion or conjugation to peptides or proteins. This was demonstrated by C3 resistant Nanofitin B10. B10 Nanofitin retains its functionality regardless of whether there is fusion at the N-terminal and C-terminal endpoints of B10 Nanofitin. This was demonstrated by the different peptide compositions and lengths and proteins (table 20). It is noted here that similar properties are observed in fusions with full length antibodies (as in WO 2019096797) or another Nanofitin (see also example 12). In addition, nanofitin can be expressed recombinantly in a variety of expression hosts (prokaryotic or eukaryotic), for example E.coli and CHO, or can be expressed by complete chemical synthesis. The biological layer interferometry experiments demonstrated that fully functional Nanofitin was recovered in all cases explored using B10.

TABLE 20 measurement of affinity of C3-binders with various tags at the N-or C-terminus.

EXAMPLE 5C 3-conjugated Nanofitin with different N-and C-terminal amino acids

The C3 conjugates described in example 3 (SEQ ID NO:25 and SEQ ID NO: 26) were produced, but lacking the N-terminal M (methionine) and/or containing K (lysine) at the C-terminus after "ERE" mode, characterized as binding to C3 and C3b and having complement inhibitory activity.

EXAMPLE 6 distribution of C3-conjugated Naofitin to brain 5 days after intrathecal infusion in rats

One of the C3-conjugated nanofittin described in example 3 or example 5 was administered to 12 male Sprague-Dawley rats (each weighing about 270 g) in a study examining the effect of continuous intrathecal infusion by an implantable Alzet mini-pump system with its cannula tip at the cranial location at the T1 vertebral level. Rats received continuous intrathecal infusion of Nanofitin (prepared in endotoxin free 1 XPBS) (0.00, 0.01 and 0.1 mg/h) by surgically implanted mini osmotic pumps for a period of 5 days. The final concentration of Nanofitin in CSF, hippocampal and hemi-brain homogenates and plasma was then assessed by LC-MS/MS on day 5. The Nanofitin administration was well tolerated, as evidenced by the lack of clinical symptoms found during the study, nor significant weight differences compared to vehicle-treated controls. The concentration of nanofitin measured in the hemi-brain or hippocampal homogenate is comparable, approximately 3-5% of the concentration measured in CSF. No Nanofitin was measured in animals treated with vehicle. Furthermore, concentrations measured in plasma were less than 0.5% of the concentration measured in CSF, indicating lower systemic exposure following intrathecal infusion. In summary, the study showed that rats had good tolerability and high brain concentrations 5 days after intrathecal pump infusion.

EXAMPLE 7 recovery of C3-bound Nanofitin by microdialysis in the brain after intrathecal infusion of rats

The Nanofittin (NF) used in example 6 was administered to 12 male Sprague-Dawley rats (each weighing about 270 g) in a study that examined the effect of continuous intrathecal infusion administered by an implantable Alzet mini-pump system with the cannula tip at the caudal location of the T13 vertebral level. Three rats per group were dosed by intrathecal infusion at a dose of 0.1mg/h for 1, 4, 24 or 36 hours. NF was measured in hippocampal cerebral interstitial fluid of microdialysis samples taken every hour within 1 to 36 hours after the start of intrathecal infusion, NF was measured from brain homogenates and plasma of the same animals at the end of the study (36 hours). At 1, 4 and 24 hours, the final NF concentrations in CSF, plasma and brain homogenates were measured with a satellite animal group. During 1 hour after the start of infusion, nanofitin was observed in the microdialysis fluid, and the measured level remained stable during the infusion period of 36 hours. The average concentration of NF in the microdialysis fluid was about 0.145. Mu.g/mL. Taken together, these data indicate that the recovery of NF from CSF to interstitial fluid is 2% to 4% after intrathecal administration in rats. The concentration in the brain is about 3% -6% of CSF. Furthermore, after intrathecal administration, the plasma concentration is at least 4-fold lower than the CSF concentration.

Example 8 at the sheathPK/PD in brain, CSF and blood 5 days after internal infusion of C3-conjugated Nanofitin

In a two-stage PK/PD and dose range discovery study, a set of 10 cynomolgus monkeys (about 3 kg) was used, with the cannula tip placed on the level of the skull at the lumbar-thoracic junction by an external piggyback continuous infusion pump system, and the effect of 5 days of continuous intrathecal infusion was examined by administering the Nanofitin (NF) used in example 6. In the A phase of the study, monkeys were infused intrathecally with vehicle, nanofitin 0.032mg/h (0.8 mg/day) or Nanofitin0.34mg/h (8 mg/day) at the lumbar-thoracic junction for 120 hours (5 days) continuously. PK and PD samples were taken from plasma and CSF samples during drug infusion and within 36 hours after pump shut down. In stage B, the same monkey as in stage A was used to intrathecally infuse the vehicle (same vehicle as received by the animal in stage A), nanofitin 0.08mg/h (2 mg/day) or Nanofitin 0.8mg/h (20 mg/day) at the lumbar-thoracic junction for 120 hours (5 days). During Nanofitin infusion, PK and PD samples were taken from plasma and CSF samples. At stage B, animals were sacrificed at 120 hours to collect brain, spinal cord and other tissues for biodistribution and pathology assessment. The pharmacokinetics of Nanofitin in plasma and CSF was assessed by LC-MS/MS. The pharmacodynamic endpoints were measured by ligand binding assays, including C3, C3 breakdown products C3a and AH50, AH50 being a measure of the functional activity of the alternative complement system in CSF and/or blood fractions. Animals were well tolerated by NF administration, as evidenced by the lack of clinical symptoms found during the study. As the dose increases, NF concentrations in CSF and plasma generally increase linearly, reaching steady state in CSF within 8-24 hours after infusion (fig. 7). CSF concentration was approximately 500 μg/mL after 5 days of infusion at a dose level of 20 mg/day. The plasma concentration of NF is about 10 to 15 times lower than in CSF. After stopping the pump infusion at stage a, NF was cleared from CSF (t 1/2 = about 3-6 hours), and t1/2 was shown in plasma for about 20 hours, consistent with what was observed after intravenous administration. The NF concentration achieved in brain homogenates was dose dependent, approximately 2-4% of the concentration in CSF (fig. 8). The NF concentration was highest in brain homogenates of brain regions of interest for the treatment of alzheimer's disease and many other neurodegenerative diseases, including the prefrontal cortex and hippocampal subzone (fig. 8). In addition, intrathecal infusion of NF at 2 mg/day or 20 mg/day dose levels resulted in lower levels of C3 decomposition product C3a in brain homogenates of the prefrontal cortex and hippocampal subzone (fig. 9), which supports the pharmacodynamic response of brain regions associated with neurodegenerative diseases.

EXAMPLE 9 plasmid-based expression of Nanofitin bound to C3 in brain cells derived from hiPSC

An expression plasmid containing a polynucleotide encoding one of the nanofitins described in example 3 or example 5 and having a signal sequence fused to the N-terminus was transfected into hiPSC-derived brain cells at a range of concentrations. Supernatants were collected at later time points and levels of Nanofitin were measured. The cells produced and secreted Nanofitin in a dose-dependent manner (fig. 10).

EXAMPLE 10 plasmid-based expression of C3-bound Nanofitin in retinal pigment epithelial cells

An expression plasmid containing a polynucleotide encoding one of the nanofitins described in example 3 or example 5 and having a signal sequence fused to the N-terminus was transfected into ARPE-19 cells (retinal pigment epithelial cell line) in a range of concentrations. Culture supernatants were collected at later time points and NF levels were measured. ARPE-19 cells produced and secreted NF in a dose-dependent manner (FIG. 11).

EXAMPLE 11 delivery of C3-bound Nanofitin Using brain shuttle

The C3 inhibitory NF described in example 3 or example 5 was coupled to the brain shuttle moiety using a linker. This demonstrates that both the brain shuttle moiety and the C3/C3b targeting moiety maintain biological activity when coupled together. Pharmacokinetic experiments in rats demonstrated that the conjugate was delivered in the brain after intravenous injection and that target brain concentrations of about 1 μg/g were reached in the brain (including in various brain regions) after systemic injection of 10mg/kg of brain shuttle-coupled C3 NF (fig. 12).

EXAMPLE 12 fusion to Albumin-bound Nanofitin prolonged half-aging of C3-bound Nanofitin Phase of time

A fusion polypeptide comprising C3 inhibitory NF (C3 NF) described in example 3 or example 5 fused to albumin-bound nanofitin (capable of binding human and rat serum albumin) was produced in E.coli (SEQ ID NO: 119). The fusion polypeptide includes a 15 amino acid peptide linker between the C-terminus of C3 inhibitory nanofitin and the N-terminus of albumin-bound nanofitin. The fusion polypeptide and the NF (His-tagged) bound to C3 were administered intravenously to rats. Blood samples were collected at various time points up to 60 hours after administration. The concentration of fusion polypeptide and C3 NF in rat plasma was measured. The measurement results showed that the half-life of the fusion polypeptide was at least 50 times greater than the half-life of C3 NF, demonstrating that the half-life of C3 inhibitory nanofitin can be significantly prolonged by fusion to albumin-bound nanofitin.

Sequence listing

<110> Ai Feiluo g Co

<120> SAC7D variants against factor C3 and medical uses thereof for treating complement mediated disorders

<130> BRV 194 - WO

<150> EP21305336.6

<151> 2021-03-18

<150> EP21305904.1

<151> 2021-06-30

<160> 119

<170> PatentIn version 3.5

<210> 1

<211> 66

<212> PRT

<213> Acidophilic sulfolobus

<400> 1

Met Val Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Val Ser Phe

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

Lys Lys

65

<210> 2

<211> 64

<212> PRT

<213> sulfolobus solfataricus

<400> 2

Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 3

<211> 65

<212> PRT

<213> Acidophilic sulfolobus

<400> 3

Met Ala Lys Val Arg Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Val Ser Phe

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Met Asp Met Leu Ala Arg Ala Glu Lys Lys

50 55 60

Lys

65

<210> 4

<211> 64

<212> PRT

<213> S.shibatae

<400> 4

Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 5

<211> 64

<212> PRT

<213> S.shibatae

<400> 5

Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 6

<211> 64

<212> PRT

<213> first Kou Dai sulfided leaf bacteria

<400> 6

Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

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

35 40 45

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

50 55 60

<210> 7

<211> 64

<212> PRT

<213> Iceltis cinerea

<400> 7

Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp

1 5 10 15

Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 8

<211> 64

<212> PRT

<213> Iceltis cinerea

<400> 8

Met Thr Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp

1 5 10 15

Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 9

<211> 64

<212> PRT

<213> Iceltis cinerea

<400> 9

Met Thr Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 10

<211> 62

<212> PRT

<213> diligence metal cocci

<400> 10

Met Ala Thr Lys Ile Lys Phe Lys Tyr Lys Gly Gln Asp Leu Glu Val

1 5 10 15

Asp Ile Ser Lys Val Lys Lys Val Trp Lys Val Gly Lys Met Val Ser

20 25 30

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

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Asn Met Ile Gly Lys Lys

50 55 60

<210> 11

<211> 62

<212> PRT

<213> Metallophylococcus cuprum

<400> 11

Met Ala Thr Lys Ile Lys Phe Lys Tyr Lys Gly Gln Asp Leu Glu Val

1 5 10 15

Asp Ile Ser Lys Val Lys Lys Val Trp Lys Val Gly Lys Met Val Ser

20 25 30

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

35 40 45

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

50 55 60

<210> 12

<211> 61

<212> PRT

<213> Haoqiao acidophilic two-sided fungus

<400> 12

Met Thr Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Leu Glu Lys Leu Glu Lys Lys

50 55 60

<210> 13

<211> 62

<212> PRT

<213> Haoqiao acidophilic two-sided fungus

<400> 13

Met Ala Thr Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val

1 5 10 15

Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser

20 25 30

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

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Asp Lys Leu Glu Lys Lys

50 55 60

<210> 14

<211> 60

<212> PRT

<213> Haoqiao acidophilic two-sided fungus

<400> 14

Met Ala Thr Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val

1 5 10 15

Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser

20 25 30

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

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55 60

<210> 15

<211> 64

<212> PRT

<213> first Kou Dai sulfided leaf bacteria

<400> 15

Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

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

35 40 45

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

50 55 60

<210> 16

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 16

Met Xaa Xaa Xaa Val Xaa Phe Lys Tyr Lys Gly Glu Glu Lys Xaa Val

1 5 10 15

Asp Xaa Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Xaa Ser

20 25 30

Phe Thr Tyr Asp Xaa Xaa Xaa Gly Lys Thr Gly Arg Gly Ala Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 17

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(10)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (34)..(34)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (42)..(42)

<223> Xaa is T or I

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (48)..(48)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 17

Met Xaa Xaa Xaa Val Xaa Phe Xaa Xaa Xaa Gly Glu Glu Lys Xaa Val

1 5 10 15

Asp Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Xaa

20 25 30

Phe Xaa Tyr Asp Xaa Xaa Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 18

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (34)..(34)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (42)..(42)

<223> Xaa is T or I

<220>

<221> variant

<222> (48)..(48)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 18

Met Xaa Xaa Xaa Val Xaa Phe Xaa Xaa Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Asp Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Leu

20 25 30

Phe Xaa Tyr Asp Xaa Xaa Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 19

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 19

Met Xaa Xaa Xaa Val Xaa Phe Xaa Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Asp Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 20

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 20

Met Xaa Xaa Xaa Val Xaa Phe Asp Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Asp Xaa Ser Lys Ile Ser Ala Val Tyr Arg Thr Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 21

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 21

Met Xaa Xaa Xaa Val Xaa Phe Ala Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Asp Xaa Ser Lys Ile Ala Asn Val Tyr Arg Val Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 22

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (40)..(40)

<223> Xaa is T or I

<220>

<221> variant

<222> (44)..(44)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is S, T, H or R

<400> 22

Met Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Thr Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Xaa Phe

20 25 30

Xaa Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 23

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (40)..(40)

<223> Xaa is T or I

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is S, T, H or R

<400> 23

Met Val Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Thr Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Leu Phe

20 25 30

Xaa Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 24

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<400> 24

Met Val Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 25

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 25

Met Val Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 26

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 26

Met Val Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 27

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (7)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (41)..(41)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (47)..(47)

<223> Xaa is S, T, H or R

<400> 27

Met Ala Thr Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Xaa Phe

20 25 30

Xaa Tyr Asp Glu Gly Gly Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 28

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (7)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<400> 28

Met Ala Thr Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Ile Leu Phe

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 29

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<400> 29

Met Ala Thr Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

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

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 30

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<400> 30

Met Ala Thr Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Asp

1 5 10 15

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

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 31

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(10)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (34)..(34)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (42)..(42)

<223> Xaa is T or I

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (48)..(48)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 31

Met Xaa Xaa Xaa Val Xaa Phe Xaa Xaa Xaa Gly Glu Glu Lys Xaa Val

1 5 10 15

Glu Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Xaa

20 25 30

Phe Xaa Tyr Asp Xaa Xaa Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 32

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (34)..(34)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (42)..(42)

<223> Xaa is T or I

<220>

<221> variant

<222> (48)..(48)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 32

Met Xaa Xaa Xaa Val Xaa Phe Xaa Xaa Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Glu Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Leu

20 25 30

Phe Xaa Tyr Asp Xaa Xaa Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 33

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 33

Met Xaa Xaa Xaa Val Xaa Phe Xaa Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Glu Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 34

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 34

Met Xaa Xaa Xaa Val Xaa Phe Asp Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Glu Xaa Ser Lys Ile Ser Ala Val Tyr Arg Thr Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 35

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 35

Met Xaa Xaa Xaa Val Xaa Phe Ala Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Glu Xaa Ser Lys Ile Ala Asn Val Tyr Arg Val Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 36

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (40)..(40)

<223> Xaa is T or I

<220>

<221> variant

<222> (44)..(44)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is S, T, H or R

<400> 36

Met Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Glu

1 5 10 15

Thr Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Xaa Phe

20 25 30

Xaa Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 37

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (40)..(40)

<223> Xaa is T or I

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is S, T, H or R

<400> 37

Met Val Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

Thr Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Leu Phe

20 25 30

Xaa Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 38

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<400> 38

Met Val Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

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

20 25 30

Ser Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 39

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 39

Met Val Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

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

20 25 30

Ser Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 40

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 40

Met Val Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

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

20 25 30

Ser Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 41

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (7)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (41)..(41)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (47)..(47)

<223> Xaa is S, T, H or R

<400> 41

Met Ala Thr Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Glu

1 5 10 15

Ile Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Xaa Phe

20 25 30

Xaa Tyr Asp Glu Gly Gly Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 42

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (7)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<400> 42

Met Ala Thr Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

Ile Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Ile Leu Phe

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 43

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<400> 43

Met Ala Thr Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

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

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 44

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<400> 44

Met Ala Thr Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Glu

1 5 10 15

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

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 45

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(10)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (17)..(17)

<223> Xaa is E or D

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (34)..(34)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (42)..(42)

<223> Xaa is T or I

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (48)..(48)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 45

Met Xaa Xaa Xaa Val Xaa Phe Xaa Xaa Xaa Gly Glu Glu Lys Xaa Val

1 5 10 15

Xaa Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Xaa

20 25 30

Phe Xaa Tyr Asp Xaa Xaa Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 46

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (17)..(17)

<223> Xaa is E or D

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (34)..(34)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (42)..(42)

<223> Xaa is T or I

<220>

<221> variant

<222> (48)..(48)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 46

Met Xaa Xaa Xaa Val Xaa Phe Xaa Xaa Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Xaa Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Leu

20 25 30

Phe Xaa Tyr Asp Xaa Xaa Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 47

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (8)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (17)..(17)

<223> Xaa is E or D

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (23)..(23)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (27)..(27)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 47

Met Xaa Xaa Xaa Val Xaa Phe Xaa Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Xaa Xaa Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 48

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (17)..(17)

<223> Xaa is E or D

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 48

Met Xaa Xaa Xaa Val Xaa Phe Asp Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Xaa Xaa Ser Lys Ile Ser Ala Val Tyr Arg Thr Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 49

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> consensus sequences

<220>

<221> variant

<222> (2)..(2)

<223> Xaa is V, A or T

<220>

<221> variant

<222> (3)..(3)

<223> Xaa is T or K

<220>

<221> variant

<222> (4)..(4)

<223> Xaa is-or K

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is R or K

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is E or Q

<220>

<221> variant

<222> (17)..(17)

<223> Xaa is E or D

<220>

<221> variant

<222> (18)..(18)

<223> Xaa is T or I

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is V or I

<220>

<221> variant

<222> (37)..(39)

<223> Xaa Xaa Xaa is EGG or DN-

<220>

<221> variant

<222> (57)..(57)

<223> Xaa is M or L

<220>

<221> variant

<222> (58)..(58)

<223> Xaa is Q, D or E

<220>

<221> variant

<222> (59)..(59)

<223> Xaa is M or K

<220>

<221> variant

<222> (61)..(68)

<223> XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa is- -K, EKS- -GKK, EK- -QKK,

ARAEREKK、ARAERE-K、ARAERE--、ARA-EKKK、E-----KK、EKS--GKK、

ACAEREKK or ACA-EKKK

<400> 49

Met Xaa Xaa Xaa Val Xaa Phe Ala Ala Thr Gly Glu Glu Lys Xaa Val

1 5 10 15

Xaa Xaa Ser Lys Ile Ala Asn Val Tyr Arg Val Gly Lys Asp Xaa Leu

20 25 30

Phe Ser Tyr Asp Xaa Xaa Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser

35 40 45

Glu Lys Asp Ala Pro Lys Glu Leu Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa

65

<210> 50

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(37)

<223> Xaa is Q or N

<220>

<221> variant

<222> (40)..(40)

<223> Xaa is T or I

<220>

<221> variant

<222> (44)..(44)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is S, T, H or R

<400> 50

Met Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

Thr Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Xaa Phe

20 25 30

Xaa Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 51

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (37)..(37)

<223> Xaa is Q or N

<220>

<221> variant

<222> (40)..(40)

<223> Xaa is T or I

<220>

<221> variant

<222> (46)..(46)

<223> Xaa is S, T, H or R

<400> 51

Met Val Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

Thr Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Leu Phe

20 25 30

Xaa Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 52

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (7)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (37)..(37)

<223> Xaa is Q or N

<400> 52

Met Val Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

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

20 25 30

Ser Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 53

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (37)..(37)

<223> Xaa is Q or N

<400> 53

Met Val Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

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

20 25 30

Ser Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 54

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (37)..(37)

<223> Xaa is Q or N

<400> 54

Met Val Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

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

20 25 30

Ser Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu

50 55 60

<210> 55

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (7)..(9)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (31)..(31)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (33)..(33)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (41)..(41)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (47)..(47)

<223> Xaa is S, T, H or R

<400> 55

Met Ala Thr Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

Ile Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Xaa Phe

20 25 30

Xaa Tyr Asp Glu Gly Gly Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 56

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (7)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (22)..(22)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (26)..(26)

<223> Xaa is S, T, V, I, L, Y or F

<400> 56

Met Ala Thr Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

Ile Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Ile Leu Phe

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 57

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<400> 57

Met Ala Thr Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

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

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 58

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sso7d that bind complement

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is E or D

<400> 58

Met Ala Thr Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Xaa

1 5 10 15

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

20 25 30

Ser Tyr Asp Glu Gly Gly Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Leu Leu Glu Lys Gln Lys Lys

50 55 60

<210> 59

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (55)..(64)

<223> XaXaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaais DMLARAEREK, DLLARAEREK or

EKLK------

<400> 59

Xaa Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Xaa Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Xaa Phe Xaa

20 25 30

Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 60

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 60

Xaa Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Xaa Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Leu Phe Xaa

20 25 30

Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 61

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 61

Xaa Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Xaa Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Xaa Leu Phe Ser

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 62

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 62

Xaa Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Xaa Xaa

1 5 10 15

Ser Lys Ile Ser Ala Val Tyr Arg Thr Gly Lys Asp Xaa Leu Phe Ser

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 63

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 63

Xaa Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Xaa Xaa

1 5 10 15

Ser Lys Ile Ala Asn Val Tyr Arg Val Gly Lys Asp Xaa Leu Phe Ser

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 64

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 64

Xaa Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Xaa Phe Xaa

20 25 30

Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 65

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 65

Xaa Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Asp Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Leu Phe Xaa

20 25 30

Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 66

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 66

Xaa Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Asp Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Xaa Leu Phe Ser

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 67

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 67

Xaa Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Asp Xaa

1 5 10 15

Ser Lys Ile Ser Ala Val Tyr Arg Thr Gly Lys Asp Xaa Leu Phe Ser

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 68

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 68

Xaa Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Asp Xaa

1 5 10 15

Ser Lys Ile Ala Asn Val Tyr Arg Val Gly Lys Asp Xaa Leu Phe Ser

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 69

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 69

Xaa Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Glu Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Xaa Phe Xaa

20 25 30

Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 70

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 70

Xaa Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Glu Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Xaa Leu Phe Xaa

20 25 30

Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 71

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 71

Xaa Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Glu Xaa

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Xaa Leu Phe Ser

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 72

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 72

Xaa Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Glu Xaa

1 5 10 15

Ser Lys Ile Ser Ala Val Tyr Arg Thr Gly Lys Asp Xaa Leu Phe Ser

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 73

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d-Aho7c that bind to complement (consensus)

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 73

Xaa Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Glu Xaa

1 5 10 15

Ser Lys Ile Ala Asn Val Tyr Arg Val Gly Lys Asp Xaa Leu Phe Ser

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 74

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (56)..(56)

<223> Xaa is M or L

<400> 74

Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Xaa Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Xaa Phe Xaa

20 25 30

Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Xaa Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 75

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<220>

<221> variant

<222> (56)..(56)

<223> Xaa is M or L

<400> 75

Val Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Xaa Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Leu Phe Xaa

20 25 30

Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Xaa Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 76

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (56)..(56)

<223> Xaa is M or L

<400> 76

Val Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Xaa Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Val Leu Phe Ser

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Xaa Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 77

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (56)..(56)

<223> Xaa is M or L

<400> 77

Val Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Xaa Thr

1 5 10 15

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

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Xaa Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 78

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (56)..(56)

<223> Xaa is M or L

<400> 78

Val Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Xaa Thr

1 5 10 15

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

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Xaa Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 79

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 79

Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Xaa Phe Xaa

20 25 30

Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 80

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 80

Val Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Asp Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Leu Phe Xaa

20 25 30

Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 81

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<400> 81

Val Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Asp Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Val Leu Phe Ser

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 82

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 82

Val Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Asp Thr

1 5 10 15

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

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 83

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 83

Val Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Asp Thr

1 5 10 15

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

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 84

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 84

Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Glu Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Xaa Phe Xaa

20 25 30

Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 85

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 85

Val Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Glu Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Val Leu Phe Xaa

20 25 30

Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 86

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<400> 86

Val Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Glu Thr

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Val Leu Phe Ser

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 87

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 87

Val Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Glu Thr

1 5 10 15

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

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 88

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> variants of Sac7d that bind to complement

<400> 88

Val Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Glu Thr

1 5 10 15

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

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 89

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 89

Thr Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Xaa Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Xaa Phe Xaa

20 25 30

Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 90

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 90

Thr Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Xaa Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Leu Phe Xaa

20 25 30

Tyr Asp Asp Xaa Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 91

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<400> 91

Thr Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Xaa Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Ile Leu Phe Ser

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 92

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<400> 92

Thr Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Xaa Ile

1 5 10 15

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

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 93

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<400> 93

Thr Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Xaa Ile

1 5 10 15

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

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 94

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 94

Thr Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Xaa Phe Xaa

20 25 30

Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 95

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 95

Thr Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Asp Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Leu Phe Xaa

20 25 30

Tyr Asp Asp Asn Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 96

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<400> 96

Thr Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Asp Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Ile Leu Phe Ser

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 97

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<400> 97

Thr Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Asp Ile

1 5 10 15

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

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 98

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<400> 98

Thr Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Asp Ile

1 5 10 15

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

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 99

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(8)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (30)..(30)

<223> Xaa is L, I, V, Y, F, M or H

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (43)..(43)

<223> Xaa is F, Y, I, V, L, M or H

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 99

Thr Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Glu Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Xaa Phe Xaa

20 25 30

Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Xaa Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 100

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(7)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<220>

<221> variant

<222> (28)..(28)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (32)..(32)

<223> Xaa is S, T or A

<220>

<221> variant

<222> (39)..(39)

<223> Xaa is T or I

<220>

<221> variant

<222> (45)..(45)

<223> Xaa is S, T, H or R

<400> 100

Thr Lys Val Lys Phe Xaa Xaa Thr Gly Glu Glu Lys Glu Val Glu Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Xaa Ile Leu Phe Xaa

20 25 30

Tyr Asp Asp Gln Gly Lys Xaa Gly Trp Gly Tyr Val Xaa Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 101

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<220>

<221> variant

<222> (6)..(6)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (20)..(20)

<223> Xaa is A, S, T, V, I, L, Y or F

<220>

<221> variant

<222> (21)..(21)

<223> Xaa is any amino acid

<220>

<221> variant

<222> (25)..(25)

<223> Xaa is S, T, V, I, L, Y or F

<400> 101

Thr Lys Val Lys Phe Xaa Ala Thr Gly Glu Glu Lys Glu Val Glu Ile

1 5 10 15

Ser Lys Ile Xaa Xaa Val Tyr Arg Xaa Gly Lys Asp Ile Leu Phe Ser

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 102

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<400> 102

Thr Lys Val Lys Phe Asp Ala Thr Gly Glu Glu Lys Glu Val Glu Ile

1 5 10 15

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

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 103

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> variants of Aho7c that bind to complement

<400> 103

Thr Lys Val Lys Phe Ala Ala Thr Gly Glu Glu Lys Glu Val Glu Ile

1 5 10 15

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

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Trp Gly Tyr Val Ser Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys

50 55

<210> 104

<211> 64

<212> PRT

<223> Artificial sequence

<220>

<223> consensus sequence of Sac7d-Aho7c

<220>

<221> variant

<222> (1)..(1)

<223> Xaa is V or T

<220>

<221> variant

<222> (16)..(16)

<223> Xaa is T or I

<220>

<221> variant

<222> (29)..(29)

<223> Xaa is V or I

<220>

<221> variant

<222> (55)..(64)

EKLK------

<400> 104

Xaa Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp Xaa

1 5 10 15

Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Xaa Ser Phe Thr

20 25 30

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

35 40 45

Ala Pro Lys Glu Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

<210> 105

<211> 6

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 105

Glu Lys Ser Gly Lys Lys

1 5

<210> 106

<211> 5

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 106

Glu Lys Gln Lys Lys

1 5

<210> 107

<211> 8

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 107

Ala Arg Ala Glu Arg Glu Lys Lys

1 5

<210> 108

<211> 7

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 108

Ala Arg Ala Glu Arg Glu Lys

1 5

<210> 109

<211> 6

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 109

Ala Arg Ala Glu Arg Glu

1 5

<210> 110

<211> 7

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 110

Ala Arg Ala Glu Lys Lys Lys

1 5

<210> 111

<211> 6

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 111

Glu Lys Ser Gly Lys Lys

1 5

<210> 112

<211> 8

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 112

Ala Cys Ala Glu Arg Glu Lys Lys

1 5

<210> 113

<211> 7

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d family consensus sequence-Table 1

<400> 113

Ala Cys Ala Glu Lys Lys Lys

1 5

<210> 114

<211> 10

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d-Aho7C consensus sequence-Table 4

<400> 114

Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

1 5 10

<210> 115

<211> 10

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d-Aho7C consensus sequence-Table 4

<400> 115

Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

1 5 10

<210> 116

<211> 4

<212> PRT

<223> Artificial sequence

<220>

<223> C-terminal of Sac7d-Aho7C consensus sequence-Table 4

<400> 116

Glu Lys Leu Lys

1

<210> 117

<211> 64

<212> PRT

<223> Artificial sequence

<220>

<223> protein F06 wherein Sac7d binds to albumin

<220>

<221> variant

<222> (15)..(15)

<223> Xaa is D or E

<220>

<221> variant

<222> (36)..(36)

<223> Xaa is N or Q

<220>

<221> variant

<222> (56)..(56)

<223> Xaa is M or L

<400> 117

Val Lys Val Lys Phe Val Phe Ser Gly Glu Glu Lys Glu Val Xaa Thr

1 5 10 15

Ser Lys Ile Lys Trp Val Leu Arg Trp Gly Lys Ala Val Gly Phe Lys

20 25 30

Tyr Asp Asp Xaa Gly Lys Ile Gly Tyr Gly Phe Val Ala Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Xaa Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 118

<211> 64

<212> PRT

<223> Artificial sequence

<220>

<223> protein F06 wherein Sac7d binds to albumin

<400> 118

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

1 5 10 15

Ser Lys Ile Lys Trp Val Leu Arg Trp Gly Lys Ala Val Gly Phe Lys

20 25 30

Tyr Asp Asp Asn Gly Lys Ile Gly Tyr Gly Phe Val Ala Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

<210> 119

<211> 64

<212> PRT

<223> Artificial sequence

<220>

<223> protein F06 wherein Sac7d binds to albumin

<400> 119

Val Lys Val Lys Phe Val Phe Ser Gly Glu Glu Lys Glu Val Glu Thr

1 5 10 15

Ser Lys Ile Lys Trp Val Leu Arg Trp Gly Lys Ala Val Gly Phe Lys

20 25 30

Tyr Asp Asp Gln Gly Lys Ile Gly Tyr Gly Phe Val Ala Glu Lys Asp

35 40 45

Ala Pro Lys Glu Leu Leu Asp Leu Leu Ala Arg Ala Glu Arg Glu Lys

50 55 60

Claims

1. A polypeptide comprising a variant of a Sac7d family member that binds to C3 and/or C3b, wherein the variant comprises 4 to 20 mutated residues in the binding interface of the Sac7d family member to its natural ligand, wherein the variant comprises a W24Y and R42W mutation, the numbering of which corresponds to the numbering of the Sac7d residues as set forth in SEQ ID No. 1.

2. The polypeptide of claim 1, further comprising at least one additional mutation selected from the group consisting of K9T, S31L and a44Y, the numbering corresponding to the numbering of the Sac7d residue as set forth in SEQ ID No. 1.

3. The polypeptide according to claim 1 or 2, further comprising at least one mutation selected from the group consisting of D16E, N37Q and M57L, the numbering corresponding to the numbering of the Sac7D residue as shown in SEQ ID No. 1.

4. A polypeptide according to any one of claims 1 to 3, wherein the mutated residue in the binding interface of the Sac7D family member with its natural ligand is selected from V2, K3, K5, K7, Y8, K9, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40, a44, S46, E47, K48, D49, a50 and P51 of Sac7D as shown in SEQ ID No. 1.

5. The polypeptide of any one of claims 1 to 4, wherein the Sac7d family member is selected from Sac7d from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), sac7e from sulfolobus acidocaldarius (Sulfolobus acidocaldarius), ach 7d from sulfolobus solfataricus (Sulfolobus solfataricus), ssh7b from sulfolobus shihei (Sulfolobus shibatae), ssh7a from sulfolobus shihei (Sulfolobus shibatae), DBP7 from sulfolobus head Kou Dai (Sulfolobus tokodaii), sis7a from sulfolobus icebergii (Sulfolobus islandicus), mse7 from metallococcus diligens (Metallosphaera sedula), mcu7 from metallococcus cuprum (Metallosphaera cuprina), ach 7a from amphibia aerophila (Acidianus hospitalis), ach 7b from amphibia aerophila (Acidianus hospitalis), ach 7c from amphibia aerophila (Acidianus hospitalis), and Sto7 from pho (4639) of rhodobacter head 4639.

6. The polypeptide of any one of claims 1 to 5, comprising SEQ ID NO 59, SEQ ID NO 45, SEQ ID NO 22, SEQ ID NO 27, SEQ ID NO 17, SEQ ID NO 64, SEQ ID NO 69, SEQ ID NO 74, SEQ ID NO 79, SEQ ID NO 84, SEQ ID NO 89, SEQ ID NO 94 or SEQ ID NO 99.

7. The polypeptide according to any one of claims 1 to 6, comprising SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 92, SEQ ID NO. 93 or amino acids 1-54 of these sequences.

8. The polypeptide of any one of claims 1 to 7, comprising a variant of a Sac7d family member that binds to C3 and/or C3 b.

9. The polypeptide of any one of claims 1 to 7, wherein the variant of a Sac7d family member that binds to C3 and/or C3b is conjugated to an organic molecule.

10. The polypeptide according to any one of claims 1 to 7, wherein the variant of the Sac7d family member that binds to C3 and/or C3b is coupled to another polypeptide, in particular another variant of the Sac7d family protein.

11. A nucleic acid molecule encoding the polypeptide of any one of claims 1 to 8 and 10.

12. A pharmaceutical composition comprising the polypeptide of any one of claims 1-10, or the nucleic acid of claim 11, and a pharmaceutically acceptable carrier.

13. The polypeptide according to any one of claims 1 to 13 or the nucleic acid according to claim 14 for use as a medicament.

14. The polypeptide according to any one of claims 1 to 13 or the nucleic acid according to claim 14 for use in the treatment of a complement-mediated disorder.

15. The polypeptide for use according to claim 14, wherein the complement-mediated disorder is selected from the group consisting of: diseases characterized by complement mediated damage to erythrocytes, in particular paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome, autoimmune diseases, in particular multiple sclerosis, diseases involving the kidney, in particular membranous proliferative glomerulonephritis, lupus nephritis, igA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G) or acute kidney injury, diseases involving the central or peripheral nervous system or neuromuscular junction, in particular neuromyelitis optica, gill-barre syndrome, multifocal motor neuropathy or myasthenia gravis, diseases involving the respiratory system, in particular pulmonary fibrosis, and diseases involving the vascular system, in particular diseases characterized by vasculitis.