WO2024235270A1 - Complement inhibitor, and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed in the present invention is a modified protein. The modified protein comprises a protein moiety and a modified moiety; the protein moiety is an OmCI mutant in which a cysteine residue is introduced by mutation; and the modified moiety is linked to the protein moiety by means of the cysteine residue introduced into the protein moiety. The modified protein of the present invention retains the original activity of the protein, and the in vivo half-life period of the protein is significantly improved, thereby laying a new material foundation for the development of drugs for treating a variety of diseases.

Description

Complement inhibitor and preparation method and use thereof Technical Field

The present invention relates to the field of biomedicine, in particular to fatty acid side chain modified OmCI mutants, and also to methods for preparing these mutants and their use in treating diseases.

Background Art

Complement is a group of globulins that are present in the blood, tissue fluid and cell membrane surface of humans and animals and have enzymatic activity after activation. Complement is a complex protein network composed of more than 50 proteins, so it is also called the complement system (Ricklin D, Lambris JD. Complement therapeutics. Semin Immunol. 2016; 28(3): 205-207). Initially, the components were named in the order of discovery of C1-C4, and then the order was corrected by the WHO in 1968 and named according to the activation order of C1-C9 (Kaufmann SH. Immunology's foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat Immunol. 2008; 9(7): 705-712.). As an important component of innate immunity, complement mainly exerts its immunological function by regulating antigens to stimulate phagocytes to remove foreign matter and damaged substances, attracting macrophages and neutrophils to cause inflammation, and activating cell-killing membrane attack complexes.

The complement-triggered immune function is achieved by a series of cascade reactions such as proteolysis to amplify the signal. There are currently three known complement activation pathways: a) the classical pathway, which requires antigen-antibody complexes to initiate C1 activation through specific immune responses; b) the alternative pathway, which does not require pathogen identification and can be initiated only by spontaneous hydrolysis or activation of C3; c) the lectin pathway, in which lectins in the blood can specifically recognize and bind to mannose on the surface of pathogenic microorganisms. This pathway can be activated by non-specific immune responses such as C3 hydrolysis or antigens in the absence of antibodies. The three pathways of complement activation are shown in Figure 1.

The three pathways are activated by different initiation factors, all of which are centered on the formation of C3 convertase and C5 convertase, which further amplify the complement response by cleaving C3 and C5 to produce corresponding biologically active fragments. In the downstream terminal pathway of the activation process, C5 is cleaved into C5a and C5b (Rawal N, Pangburn MK. Structure/function of C5 convertases of complement. Int Immunopharmacol. 2001; 1(3): 415-422). C5a is an anaphylatoxin and an important chemotactic protein that plays a key role in recruiting inflammatory cells. C5b recruits the membrane attack complex (MAC) composed of C6, C7, C8 and C9. As the end product of the complement cascade reaction, MAC has the function of dissolving cell membranes. It can bind to the cell membrane of pathogens to form a hole, namely a transmembrane channel, causing penetration of target cells, thereby killing or destroying pathogen cells (Serna M, Giles JL, Morgan BP, Bubeck D. Structural basis of complement membrane attack complex formation. Nat Commun. 2016; 7:10587).

The immunological function of complement activation has a positive impact on the body's self-protection and anti-infection effects, but the damage and dysregulation of complement response are considered to be important pathogenic factors for some autoimmune diseases and various inflammatory diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD) and many other diseases. Complement C5, as a representative molecule of the membrane attack complex in the complement system, is a large protein of 188 kDa with a concentration of 75 μg/ml in serum. Targeting this protein can regulate the activation of all three different pathways of complement. Body signal.

A natural protein with a molecular weight of about 17 kDa was extracted from the salivary glands of the soft tick (Ornithodoros moubata). Studies have shown that this protein can specifically bind to complement C5, inhibit the activation pathway of complement downstream, and prevent the formation of MAC. In addition to targeting complement C5, studies have also shown that OmCI protein has the ability to inhibit the activity of leukotriene B4 (LTB4), providing additional anti-inflammatory function (Roversi P, Ryffel B, Togbe D, et al. Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury. J Biol Chem. 2013; 288(26): 18789-18802). Therefore, OmCI has a variety of clinical application potentials.

Rapid renal clearance of protein and peptide drugs is a common phenomenon in the development of biopharmaceuticals. Although the half-life of OmCI is longer than that of other small molecule biologics, and the circulation half-life of pOmCI is 30h, its clinical use still requires daily intravenous administration, which inevitably leads to poor patient compliance.

Among the several strategies currently used to extend the half-life of protein peptides in vivo, fatty acid modification technology is a long-acting strategy based on endogenous albumin. Human serum albumin (HSA), as the most important protein in human plasma, can be used as a carrier to transport drugs, metabolites and fatty acids, and its half-life in the human body is about 19 days. Fatty acids reversibly bind to human serum albumin (HAS) in vivo through non-covalent bonds and participate in the circulation mediated by the neonatal receptor (FcRn), thereby achieving the long-acting effect of the drug. Currently, this strategy has also been widely used in a variety of peptide drugs. For example, Semaglutide, developed by Novo Nordisk, is based on human GLP-1 and is modified with fatty acid side chains to extend its injection frequency to once a week (Lau J, Bloch P, L, et al. Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J Med Chem. 2015; 58(18):7370-7380).

However, there is no prior art method for applying fatty acid modification strategy to OmCI protein to extend its half-life in vivo.

Summary of the invention

The object of the present invention is to provide a method for extending the in vivo half-life of an OmCI protein, as well as an OmCI protein with an extended half-life and a pharmaceutical composition containing the same.

In a first aspect, the present invention provides a modified protein, comprising a protein portion and a modification portion, wherein a cysteine residue is introduced into the protein portion by mutation, and the modification portion is connected to the protein portion via the cysteine residue introduced into the protein portion, and the protein is OmCI or an active fragment thereof.

In a preferred embodiment, the mutation is a substitution or an insertion; preferably a substitution.

In a preferred embodiment, the OmCI has the amino acid sequence shown in SEQ ID NO:1.

In a preferred embodiment, the OmCI has the amino acid sequence shown in SEQ ID NO:1, and T90, K95, T97, E126 or S156 is mutated to a cysteine residue.

In a preferred embodiment, the amino acid sequence of OmCI is as shown in any one of SEQ ID NO:4-8.

In a preferred embodiment, the amino acid sequence of OmCI is as shown in any one of SEQ ID NO: 4, 5, 7, and 8.

In a preferred embodiment, the modified moiety is capable of extending the in vivo half-life of the protein moiety.

In a preferred embodiment, the modified portion is capable of binding specifically or non-specifically to a protein in plasma.

In a preferred embodiment, the protein in the plasma has a long half-life; for example, the half-life of the protein in the plasma is 10 days or more, preferably 15 days or more, more preferably 19 days or more.

In a preferred embodiment, the protein in the plasma is human serum albumin.

In a preferred embodiment, the modifying moiety is an albumin binder.

In a preferred embodiment, the albumin binder is a fatty acid.

In a preferred embodiment, the structure of the albumin binder is shown below:

[R ₁ -(X ₁ ) _m -(X ₂ ) _n -K] _o -X ₃ -(ε-R ₂ );

In the formula, K is lysine

_R1 is a substituted or unsubstituted _C1-20 acyl group;

m is an integer from 0 to 5;

n is an integer from 0 to 5;

o is an integer of 1-3 (preferably 1 or 2);

_X1 and _X2 are independently selected from the following group: alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), tranexamic acid (Trx), 8-amino-3,6-dioxaoctanoic acid (AEEA), PEG;

X ₃ is absent or is a linking moiety of the following formula:

(R ₃ ) _o R ₄ R ₅ ; wherein R ₃ is a substituted or unsubstituted C _1-3 acyl group, R ₄ is a C _5-8 aryl or heteroaryl group, and R ₅ is a substituted or unsubstituted amino group;

_R2 is selected from halogen-substituted _C1-6 acyl.

In a preferred embodiment, R ₁ is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, and 11-carboxyundecanoyl.

In a preferred embodiment, R ₁ is selected from the group consisting of lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, and 19-carboxynonadecanoyl.

In a preferred embodiment, m is an integer of 1-3.

In a preferred embodiment, n is an integer of 1-3.

In a preferred embodiment, _X1 and _X2 are independently selected from the following group: 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), β-alanine (β-Ala), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), γ-glutamic acid (γ-Glu), glycine (Gly), serine (Ser), tyrosine (Tyr), 8-amino-3,6-dioxaoctanoic acid (AEEA), lysine (Lys).

In a preferred embodiment, _X1 and _X2 are independently selected from the group consisting of glutamic acid (Glu), γ-glutamic acid (γ-Glu), 8-amino-3,6-dioxaoctanoic acid (AEEA), and lysine (Lys).

In a preferred embodiment, the amino acid is preferably an L-amino acid.

In a preferred embodiment, R ₃ is formyl, R ₄ is phenyl, and R ₅ is amino.

In a preferred embodiment, R ₂ is selected from halogen-substituted C _1-3 acyl.

In a preferred embodiment, R ₂ is selected from iodoacetyl or bromoacetyl; more preferably bromoacetyl.

In a preferred embodiment, the albumin binder is selected from the group consisting of:

In a preferred embodiment, it is characterized in that the modified protein is selected from the following group:

In a second aspect, the present invention provides an OmCI mutant having an amino acid sequence as shown in SEQ ID NO: 1, and T90, K95, T97, E126 or S156 are mutated to cysteine residues.

In a preferred embodiment, the amino acid sequence of OmCI is as shown in any one of SEQ ID NO:4-8.

In a preferred embodiment, the amino acid sequence of OmCI is as shown in any one of SEQ ID NO: 4, 5, 7, and 8.

In a third aspect, the present invention provides a pharmaceutical composition comprising the modified protein according to the first aspect or the OmCI mutant according to the second aspect and a pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutical composition is used as a complement inhibitor.

In a preferred embodiment, the pharmaceutical composition is suitable for use in autoimmune diseases, inflammation, leukotriene or hydroxyeicosanoid mediated diseases.

In a preferred embodiment, the pharmaceutical composition is suitable for paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), respiratory viral infections, autoimmune bullous diseases, and eye diseases (e.g., proliferative retinal diseases).

In a fourth aspect, the present invention provides use of the modified protein of the first aspect or the OmCI mutant of the second aspect in preparing a drug.

In a preferred embodiment, the drug is a complement inhibitor.

In a preferred embodiment, the medicament is suitable for autoimmune diseases, inflammation, leukotriene or hydroxyeicosanoid mediated diseases.

In a preferred embodiment, the medicament is suitable for paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), respiratory viral infections, autoimmune blistering diseases, eye diseases (e.g., proliferative retinal diseases).

In a fifth aspect, the present invention provides an albumin binder, the structure of which is shown in the following formula:

[R ₁ -(X ₁ ) _m -(X ₂ ) _n -K] _o -X ₃ -(ε-R ₂ );

In the formula, K is lysine

_R1 is a substituted or unsubstituted _C1-20 acyl group;

m is an integer from 0 to 5;

n is an integer from 0 to 5;

o is an integer of 1-3 (preferably 1 or 2);

_X1 and _X2 are independently selected from the following group: alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asp), Asn, Cysteine (Cys), Glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), Glutamine (Gln), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Proline (Pro), Phenylalanine (Phe), Serine (Ser), Tyrosine (Tyr), Threonine (Thr), Tryptophan (Trp), Valine (Val), Methionine (Met), Tranexamic acid (Trx), 8-amino-3,6-dioxaoctanoic acid (AEEA), PEG;

X ₃ is absent or is a linking moiety of the following formula:

(R ₃ ) _o R ₄ R ₅ ; wherein R ₃ is a substituted or unsubstituted C _1-3 acyl group, R ₄ is a C _5-8 aryl or heteroaryl group, and R ₅ is a substituted or unsubstituted amino group;

_R2 is selected from halogen-substituted _C1-6 acyl.

In a preferred embodiment, R ₁ is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, and 11-carboxyundecanoyl.

In a preferred embodiment, R ₁ is selected from the group consisting of lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, and 19-carboxynonadecanoyl.

In a preferred embodiment, m is an integer of 1-3.

In a preferred embodiment, n is an integer of 1-3.

In a preferred embodiment, _X1 and _X2 are independently selected from the following group: 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), β-alanine (β-Ala), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), γ-glutamic acid (γ-Glu), glycine (Gly), serine (Ser), tyrosine (Tyr), 8-amino-3,6-dioxaoctanoic acid (AEEA), lysine (Lys).

In a preferred embodiment, _X1 and _X2 are independently selected from the group consisting of glutamic acid (Glu), γ-glutamic acid (γ-Glu), 8-amino-3,6-dioxaoctanoic acid (AEEA), and lysine (Lys).

In a preferred embodiment, the amino acid is preferably an L-amino acid.

In a preferred embodiment, R ₃ is formyl, R ₄ is phenyl, and R ₅ is amino.

In a preferred embodiment, R ₂ is selected from halogen-substituted C _1-3 acyl.

In a preferred embodiment, R ₂ is selected from iodoacetyl or bromoacetyl; more preferably bromoacetyl.

In a specific embodiment, the albumin binder is selected from the group consisting of:

In a sixth aspect, the present invention provides an isolated nucleic acid molecule encoding the OmCI mutant of the second aspect.

In a seventh aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the sixth aspect.

In an eighth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector described in the seventh aspect or the nucleic acid molecule described in the sixth aspect is integrated into the genome of the host cell.

In a ninth aspect, the present invention provides use of the OmCI mutant described in the second aspect in preparing the modified protein described in the first aspect.

In a tenth aspect, the present invention provides a therapeutic method, comprising the step of administering a therapeutically effective amount of the modified protein of the first aspect, the OmCI mutant of the second aspect, or the pharmaceutical composition of the third aspect to a subject in need thereof.

In preferred embodiments, the treatment methods are applicable to autoimmune diseases, inflammation, leukotriene or hydroxyeicosanoid mediated diseases.

In a preferred embodiment, the treatment method is suitable for paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), respiratory viral infections, autoimmune blistering diseases, and eye diseases (e.g., proliferative retinal diseases).

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the three pathways of complement activation;

Figure 2 shows the SDS-PAGE images of the purified recombinant OmCI protein and its four mutants described in Examples 1 and 2; wherein, "1" is OmCIT90C; "2" is OmCIK95C; "3" is OmCIT97C; "4" is OmCIS156C; "5" is OmCI; "M" is Marker; "6" is OmCIT90C+DTT; "7" is OmCIK95C+DTT; "8" is OmCIT97C+DTT; "9" is OmCIS156C+DTT; and "10" is OmCI+DTT;

FIG3 shows the inhibition of PNH-like erythrocyte hemolysis in vitro by the recombinant OmCI protein and two modified derivatives described in Example 8;

FIG. 4 shows the pharmacokinetic parameters of the recombinant OmCI protein and two modified derivatives described in Example 11 in mice.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventor unexpectedly discovered a complement inhibitor targeting C5, an OmCI mutant modified derivative. The OmCI mutant modified derivative has good complement C5 inhibitory activity and excellent in vivo half-life, thereby laying a new material foundation for the development of related drugs and having great clinical development potential. On this basis, the present invention was completed.

definition

Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this article, it is intended to refer to the corresponding commodity or its active ingredient.

The term "isolated nucleic acid molecule" refers to a nucleic acid molecule provided herein that is: 1) separated from at least about 50% of the proteins, lipids, carbohydrates or other materials that coexist with the nucleic acid when the nucleic acid is separated from the source cell, 2) not connected to all or part of a polynucleotide that is naturally connected to the "isolated nucleic acid molecule", 3) effectively connected to a polynucleotide that is not naturally connected to it, or 4) not naturally present as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other pollutants that may interfere with its use in polypeptide production or its treatment, diagnosis, prevention or research purposes that exist in its natural environment.

The term "vector" is used to refer to any molecule (eg, nucleic acid, plasmid or virus) used to transfer coding information to a host cell.

The term "expression vector" refers to a vector suitable for host cell transformation and containing a nucleic acid sequence that directs and/or controls the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, transcription, translation, and RNA splicing (if introns are present). process.

The term "operably linked" is used herein to refer to an arrangement of flanking sequences, wherein the flanking sequences described herein are configured or assembled so as to perform their conventional functions. Thus, a flanking sequence that is operably linked to a coding sequence may be capable of effecting replication, transcription, and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of the coding sequence. A flanking sequence need not be contiguous with a coding sequence, as long as it functions properly. Thus, for example, an intervening non-translated but transcribed sequence may be present between a promoter sequence and a coding sequence, and the promoter sequence may still be considered to be "operably linked" to a coding sequence.

The term "host cell" is used to refer to a cell that is transformed or capable of being transformed by a nucleic acid sequence (e.g., a nucleic acid provided herein) and then capable of expressing a selected target gene. The term includes progeny of a parent cell, whether or not the progeny is identical to the original parent in morphology or genetic composition, as long as the selected gene is present.

"Substantially" means almost completely or completely, such as satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or greater, and 95% or greater. As used herein, "sequence identity" is determined by aligning the sequence of a reference DNA with another DNA sequence, thereby maximizing the overlap between the two sequences and simultaneously minimizing sequence gaps, wherein any protruding sequences between the two sequences are ignored. For any sequence identity described herein, at least 80% is preferred, 85% is more preferred, 90% is more preferred, 95% sequence identity is still more preferred, and 96%, 97%, 98% and 99% sequence identity are most preferred.

The abbreviations for the amino acid residues in proteins are as follows: Phe or F for phenylalanine; Leu or L for leucine; Ile or I for isoleucine; Met or M for methionine; Val or V for valine; Ser or S for serine; Pro or P for proline; Thr or T for threonine; Ala or A for alanine; Tyrosine is Tyr or Y; His or H for histidine; Gln or Q for glutamine; Asn or N for asparagine; Lys or Lys; Aspartic acid is Asp or D; Glu or E for glutamate; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G.

The term "optionally" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes both the occurrence of the event or situation and the non-occurrence of the event or situation. For example, an ethyl group is "optionally" substituted with a halogen, which means that the ethyl group may be unsubstituted (CH ₂ CH ₃ ), monosubstituted (such as CH ₂ CH ₂ F), polysubstituted (such as CHFCH ₂ F, CH ₂ CHF _2, etc.) or fully substituted (CF ₂ CF ₃ ). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will be introduced.

As used herein, Cm-n means that there are m-n carbon atoms in the moiety. For example, "C0-6 alkylene" means that the alkylene has 0-6 carbon atoms, and when the alkylene has 0 carbon atoms, the group is a bond.

Numeric ranges herein refer to each integer in the given range. For example, "C1-6" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 2 R's, each R has an independent choice.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, as long as the particular atom The valence state is normal and the substituted compound is stable. When the substituent is oxo (ie, =O), it means that two hydrogen atoms are replaced, and oxo will not occur on the aromatic group.

The term "derivative" as a noun refers to a modified protein, i.e. a protein to which a moiety is bonded in order to modify the properties of the protein; as a verb, the term refers to the process of bonding a moiety to a protein to modify the properties of the protein.

OmCI and mutants thereof of the present invention

In the context of the present invention, the term "OmCI" used herein means a recombinant C5 complement inhibitor derived from ticks, which is a natural protein with a molecular weight of about 17 kDa extracted from the salivary glands of soft ticks (Ornithodoros moubata). Studies have found that the protein can specifically bind to complement C5, inhibit the activation pathway downstream of complement, and prevent the formation of MAC. CN1798841B discloses the recombinant sequence of the complement inhibitor. The inhibitor sequence consists of amino acids 1 to 168, wherein the first 18 amino acids of the protein sequence form a signal sequence, and its mature form is a protein composed of amino acid sequences 19 to 168 shown in CN1798841B, and it is named "OmCI protein", "Coversin" or "Nomacopan". The OmCI protein structure belongs to the lipocalin family. It has a compact folded structure, including a central eight-stranded antiparallel β barrel with three pairs of disulfide bonds at positions 24 and 146, 118 and 147, and 56 and 168, respectively (Roversi P, Lissina O, Johnson S, et al. The structure of OMCI, a novel lipocalin inhibitor of the complement system. J Mol Biol. 2007; 369(3): 784-793). In addition to its targeted binding to complement C5, studies have also shown that OmCI protein has the ability to inhibit the activity of leukotriene B4 (LTB4), providing additional anti-inflammatory function (Roversi P, Ryffel B, Togbe D, et al. Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury. J Biol Chem. 2013; 288(26): 18789-18802).

OmCI has a variety of clinical application potentials. For example, CN102066412A discloses its ability to bind to the eicosanoids of LTB4 and its use in treating diseases mediated by leukotrienes or hydroxy eicosanoids; CN101340926A discloses its effect in treating myasthenia gravis; CN102762223B discloses its effect in preventing respiratory viral infections and inflammation; CN106659767B discloses that it can treat or prevent subjects with C5 polymorphism and complement-mediated disorders; CN110896606A discloses that it can be used as an effective method for treating and preventing autoimmune bullous disease (AIBD); CN110831617A discloses that it can be used to treat eye diseases; CN114072206A discloses that it can treat or prevent proliferative retinal diseases.

The term mutant as used herein has the meaning conventionally understood by those skilled in the art. In a specific embodiment, a mutant is understood to be a compound obtained by the following steps: replacing one or more amino acid residues in the OmCI sequence with another natural or non-natural amino acid; and/or adding one or more natural or non-natural amino acids to the OmCI sequence; and/or deleting one or more amino acid residues from the OmCI sequence, wherein any of these steps may be optionally followed by further derivatization of one or more amino acid residues. In particular, in the case where an amino acid residue is substituted by another amino acid residue from the same group (i.e., substituted by another amino acid residue with similar properties), such substitutions are conservative. Amino acids can be suitably divided into the following groups based on their properties: basic amino acids (e.g., arginine, lysine, histidine), acidic amino acids (e.g., glutamic acid and aspartic acid), polar amino acids (e.g., glutamine, cysteine and asparagine), hydrophobic amino acids (e.g., leucine, isoleucine, proline, methionine and valine), aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine) and small amino acids (e.g., glycine, alanine, serine and threonine). Typically, the OmCI mutant has at least 80%, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the native OmCI from ticks.

As used herein, the term "OmCI fusion protein" refers to the fusion of one or more amino acid residues (eg, a heterologous protein or peptide) to the N-terminus or C-terminus of any of the OmCI mutants described herein. Heterologous peptides and polypeptides include, but are not limited to, epitopes for detecting and/or isolating OmCI protein mutants: transmembrane receptor proteins or portions thereof, such as extracellular domains or transmembrane and intracellular domains; ligands or portions thereof that bind to transmembrane receptor proteins; enzymes or catalytically active portions thereof; polypeptides or peptides that promote oligomerization, such as leucine zipper domains; polypeptides or peptides that improve stability, such as immunoglobulin constant regions (e.g., domains); half-life extension sequences comprising a combination of two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) naturally occurring or non-naturally occurring, charged and/or uncharged amino acids (e.g., serine, glycine, glutamic acid, or aspartic acid) designed to form a fusion partner of a predominantly hydrophilic or predominantly hydrophobic mutant; functional or non-functional native antibodies, or heavy or light chains thereof; and polypeptides having, for example, therapeutic activity, other than the OmCI mutants of the present invention. OmCI fusion proteins can be prepared by fusing heterologous sequences to the N-terminus or C-terminus of OmCI polypeptide mutants. The heterologous sequence described herein can be an amino acid sequence or a polymer containing non-amino acids. The heterologous sequence can be directly fused to the OmCI mutant or fused through a joint or an adapter molecule. The joint or adapter molecule can be one or more amino acid residues (or amino acid polymers), such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues (or amino acid polymers), preferably 10-50 amino acid residues (or amino acid polymers), such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 residues (or amino acid polymers), more preferably 15-35 amino acid residues (or amino acid polymers). The joint or adapter molecule can also be designed to have a cleavage site for DNA restriction endonucleases or proteases for separating the fusion part.

The terms "analogs" or "derivatives" before and after OmCI as referred to herein, i.e., OmCI analogs or OmCI derivatives, refer to proteins that are or can be derived from natural OmCI, in particular, from or can be derived from SEQ ID NO: 1, i.e., through the modification of its amino acid sequence. Such modification, amendment or change may include substitution, deletion and/or addition of one or more amino acids. For example, amino acids may be added and/or deleted at the C-terminus, N-terminus or inside the amino acid sequence. Preferably, amino acids are added and/or deleted at the C-terminus and/or N-terminus, more preferably at the N-terminus. An amino acid sequence with amino acids deleted at the C-terminus or N-terminus may also be referred to as a truncated sequence, which is known in the art. Similarly, amino acids added inside the sequence may be referred to as insertions.

Rapid renal clearance of protein and peptide drugs is a common phenomenon in the development of biopharmaceuticals. The circulation half-life of OmCI is 30h, and daily administration is required clinically, which will inevitably affect the compliance of patients. In the present invention, the inventors use albumin binders to modify OmCI to extend its half-life. In this article, albumin binders or albumin binding fragments have the same meaning. The meaning of the two themselves is not restrictive, but descriptive, because it reflects the overall goal or purpose of being connected to OmCI, that is, the resulting derivative (or analog) can be combined with human serum albumin, and the purpose of the combination is to provide or at least contribute to the persistence effect of the analog of the present invention. If necessary, the term can also be replaced by other general chemical terms such as compounds.

In the formula of the present invention, the albumin binder is a fatty acid. In a specific embodiment, the structure of the albumin binder is shown in the following formula:

[R ₁ -(X ₁ ) _m -(X ₂ ) _n -K] _o -X ₃ -(ε-R ₂ );

wherein K, R ₁ , m, n, o, X ₁ , X ₂ and X ₃ are as defined above.

In a specific embodiment, OmCI and its mutants are prepared as follows:

In one or more examples herein, methods for preparing recombinant OmCI and OmCI mutants are provided. OmCI and its mutants can be expressed in bacteria, such as E. coli, mammals, yeast, such as Pichia pastoris, and plant expression systems. Expression can be performed by exogenous expression (when the host cell naturally contains the desired genetic code) or by endogenous expression. In some embodiments, OmCI and its mutants are expressed in E. coli.

Although recombinant-based methods for preparing proteins can vary, recombinant methods typically involve constructing a nucleic acid encoding a desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell, and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic host cells are known to those of ordinary skill in the art.

In one or more embodiments of the present invention, in order to obtain an OmCI analog of the correct sequence, a nucleotide sequence encoding an acid-sensitive tag can be inserted or added to the encoded sequence in a frame-matched manner, thereby producing a fusion protein comprising a desired polypeptide and a polypeptide containing an acid-sensitive tag.

In some embodiments, a method using genetic engineering is provided to artificially synthesize and express a fusion protein (precursor protein) comprising a desired polypeptide and a tag polypeptide, insert the recombinant gene sequence into an expression vector, transform host cells, and prepare OmCI and its mutants by fermentation, high-pressure cell disruption, enzyme digestion, purification, etc. The purity of OmCI and its mutants can be determined by any of a variety of known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc., and its molecular weight can be identified by mass spectrometry.

In the present invention, OmCI cysteine mutants were prepared as follows:

In one or more embodiments of the present invention, rationally designed OmCI mutants are provided, in particular, mutants in which certain amino acids of OmCI are mutated to cysteine. Including, cysteine mutations were performed on T90, K95, T97, E126 and S156 based on the sequence of SEQ NO:1, respectively, to obtain five OmCI cysteine mutants with sequences of T90C (SEQ NO:4), K95C (SEQ NO:5), T97C (SEQ NO:6), E126C (SEQ NO:7) and S158C (SEQ NO:8). Four OmCI cysteine mutants including T90C (SEQ NO:4), K95C (SEQ NO:5), T97C (SEQ NO:7) and S158C (SEQ NO:8) were successfully prepared and their activities were determined by the classical pathway hemolytic test. As is well known to those skilled in the art, compounds linked by disulfide bonds, including but not limited to OmCI-Cys, OmCI-reduced glutathione, OmCI-cysteamine and the like obtained by reacting with free Cys of the OmCI mutant are also included in the OmCI mutant.

In the present invention, the albumin binder of the present invention is prepared as follows:

In some embodiments, it is mentioned that the solid phase synthesis of the albumin binding fragment can be carried out according to conventional solid phase synthesis methods. The albumin binding fragment can be purified by various known methods, and the purity and molecular weight of the albumin binding fragment can be verified by high performance liquid chromatography and mass spectrometry analysis.

The derivatives generally comprise an albumin binding fragment, the tag being non-toxic, non-naturally occurring and biocompatible, typically the albumin binding fragment having a binding affinity for human serum albumin of less than about 10 μM or even less than about 1 μM.

In the present invention, the OmCI mutant modified derivatives can be prepared as follows:

In one or more embodiments herein, methods for preparing OmCI modified derivatives are provided. The preparation of OmCI modified derivatives as described herein can be purified by techniques known in the art, such as reversed phase high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a specific protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. Ion exchange chromatography can be used to separate the modified derivatives, substantially as described in the embodiments. The purity of the modified derivatives can be determined by any of a variety of known analytical methods, including gel electrophoresis, reversed phase high performance liquid chromatography, etc., and the molecular weight is verified by mass spectrometry.

OmCI mutant derivatives with albumin binder modifications can be prepared as follows:

In one or more embodiments herein, the albumin binding fragment reacts with free cysteine on the OmCI mutant through a halogenation reaction, ie, the fatty acid is modified to obtain an OmCI derivative.

In the context of the present invention, the term "pharmaceutically acceptable salt" means a salt that is harmless to the patient. The salt includes pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. Acid addition salts include salts of inorganic acids and organic acids. Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, etc. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, tartaric acid, ascorbic acid, pamoic acid, dimethoxy salicylic acid, ethanedisulfonic acid, gluconic acid, citric acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. In addition, metal salts include lithium salts, sodium salts, potassium salts, magnesium salts, etc. Ammonium salts and alkylated ammonium salts include ammonium salts, methylammonium salts, dimethylammonium salts, trimethylammonium salts, ethylammonium salts, hydroxyethylammonium salts, diethylammonium salts, butylammonium salts, tetramethylammonium salts, etc.

The present application also includes isotope-labeled compounds of the present application that are identical to those described herein, but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I, and 36Cl, etc., respectively.

Certain isotopically labeled compounds of the present invention (e.g., those labeled with 3H and 14C) can be used in compound and/or substrate tissue distribution analysis. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. In addition, substitution with heavier isotopes (such as deuterium (i.e., 2H)) can provide certain therapeutic advantages (e.g., increased in vivo half-life or reduced dosage requirements) produced by higher metabolic stability, and therefore may be preferred in some cases. Positron emitting isotopes, such as 15O, 13N, 11C, and 18F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present invention can generally be prepared by replacing an isotopically labeled reagent with an isotopically labeled reagent by the following procedures similar to those disclosed in the schemes and/or embodiments below.

Therapeutic uses of OmCI analogs: OmCI analogs can be used to treat, diagnose, ameliorate or prevent a variety of diseases, disorders or conditions, including but not limited to immunotherapy. Pemphigus, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD) and many other diseases.

In use, the OmCI analogs described herein can be administered to patients in need of the treatment of PNH or aHUS and other diseases or conditions. Administration can be as described herein, for example, by intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, or oral administration in the form of tablets or liquid preparations. In most cases, the required dose can be determined by clinical staff as described herein, and can represent a therapeutically effective dose of OmCI protein analogs. It is obvious to those skilled in the art that the therapeutically effective dose of OmCI analogs will depend on, among other things, the dosing regimen, the unit dose of the substance administered (whether or not the nucleic acid molecule or polypeptide is administered in combination with other therapeutic agents), the immune status and the health status of the recipient. The term "therapeutically effective dose" as used herein means the amount of OmCI analogs that elicits a biological or drug response in a tissue system, animal or human that is sought by a researcher or other clinician, including the alleviation of the symptoms of the disease or condition to be treated.

Advantages of the present invention:

1. The present invention provides a new modified protein;

2. The modified protein of the present invention retains the original activity of the protein, and its half-life in vivo is also significantly improved;

3. The preparation process of the modified protein of the present invention is simple and can be easily obtained by biotechnology, chemical synthesis technology and other technical means; and

4. The modified protein of the present invention lays a new material foundation for the development of therapeutic drugs for various diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical bullous pemphigoid, atopic keratoconjunctivitis, thrombotic microangiopathy (TMA), myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, age-related macular degeneration (AMD), etc.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

Example

List of abbreviations
SDS: Sodium dodecyl sulfate
DTT: dithiothreitol
TFA: trifluoroacetic acid
TCEP: Tris(2-carboxyethyl)phosphine
AEEA: 2-(2-(2-aminoethoxy)ethoxy)acetic acid
RP-HPLC: Reversed-Phase High-Performance Liquid Chromatography
Tris-HCl: tris(hydroxymethyl)aminomethane hydrochloride;
Alloc: allyloxycarbonyl;
Fmoc: 9-fluorenylmethoxycarbonyl;
DIEA: N,N-diisopropylethylamine;
DCM: dichloromethane;
DIC: N,N'-diisopropylcarbodiimide;
HOBt: 1-hydroxybenzotriazole;
DMF: N,N-dimethylformamide;
PIP: piperidine;
IPTG: isopropyl-β-D-thiogalactoside;
TCEP: tris(2-carboxyethyl)phosphine;
Tris: tris(hydroxymethyl)aminomethane;
GABA: gamma-aminobutyric acid;
Fmoc-Lys(Alloc)-OH: Nα-(9-fluorenylmethoxycarbonyl)-Nε-allyloxycarbonyl-L-lysine;
Fmoc-AEEA-OH: [2-[2-(9-Fluorenylmethoxycarbonyl-amino)ethoxy]ethoxy]acetic acid;
Fmoc-Glu-OtBu: N-Fluorenylmethoxycarbonyl-L-glutamic acid-α-tert-butyl ester;
PBS: potassium dihydrogen phosphate (KH ₂ PO ₄ ): 0.24 g/L, disodium hydrogen phosphate (Na ₂ HPO ₄ ): 1.44 g/L, sodium chloride
(NaCl): 8g/L, potassium chloride (KCl): 0.2g/L, pH 7.4.

General Detection and Characterization Methods

RP-HPLC

LC-MS method

SDS-PAGE analysis

A 15% precast gel was used. The samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were prepared, loaded onto the gel and electrophoresis was performed as described by the manufacturer.

Protein quantification

The protein concentration was determined by using a BCA kit.

Data were analyzed using GraphPad Prims version 8.01 (GraphPad Software Inc., San Diego, CA, USA).

Example 1: Preparation of recombinant OmCI protein

(1) OmCI is naturally derived from the salivary glands of ticks, and its sequence is available on Uniport (ID: Q5YD09), such as SEQ ID NO: 1. The natural OmCI protein consists of amino acids 1 to 168, of which the first 18 amino acids of the protein sequence form a signal sequence that is not essential for C5 binding activity or for LTB4 binding activity. The mature form of the amino acid sequence 19 to 168 constitutes the protein, so the amino acid sequence of the mature protein can be used for the efficiency of recombinant protein production.

Considering the three pairs of disulfide bonds in the protein structure, an extracellular expression system of recombinant OmCI protein was established in the form of fusion protein. An expression cassette containing an N-terminal enterokinase recognition site (GSGDEGD) and a periplasmic binding protein (ArgT; UniProt ID P09551) for OmCI expression was constructed and inserted into the pET30 plasmid. Then it was transformed into competent E. coli BL21 (DE3) bacteria, and an engineered strain for expressing the ArgT-GSGDEGD-OmCI (SEQ ID NO: 2) fusion protein was obtained by screening. After high-density fermentation, the engineered bacteria expressed the ArgT-GSGDEGD-OmCI fusion protein in a soluble form outside the cell.

(2) Soluble fusion protein ArgT-GSGDEGD-OmCI (SEQ ID NO: 2), after enzyme cleavage, purified to obtain soluble target OmCI recombinant protein. First, the pH of the fermentation broth was adjusted to 4.0, and the broth was clarified after centrifugation. The pH was then adjusted to 8.0, and recombinant bovine enterokinase light chain (which can specifically cleave the GSGDEGD site) was added at 37°C for enzyme cleavage reaction. The pH of the treated enzyme cleavage solution was adjusted to 6.5, and anion exchange chromatography (HiTrap Q HP (5 ml, purchased from Cytiva) was used to maintain the chromatography pH at 6.5. The elution was performed with a linear salt gradient of 0-500 mM NaCl. After SDS-PAGE analysis and RP-HPLC analysis, the soluble target OmCI protein with high purity was obtained. Finally, the OmCI protein was concentrated by ultrafiltration using a MWCO 3kDa filter membrane and replaced in PBS.

After LC-MS analysis, the measured molecular weight of OmCI prepared according to the above method was 16779.85 Da, which was consistent with the theoretical molecular weight (16779.59 Da).

Example 2: Preparation of OmCI mutant protein

(1) Design of OmCI mutant proteins

Based on the crystal structure of OmCI and related receptor complexes, a single Cys mutation site was designed using bioinformatics software. The purpose was to obtain free Cys that could be used for chemical modification of the albumin binding fragment. First, the crystal structure of OmCI (PDB ID: 2CM4) and the complex structures of OmCI with the receptor (complement C5) and the receptor (LTB4) (OmCI-C5; PDB ID: 5HCC and OmCI-LTB4; PDB ID: 3ZUO) were downloaded from the Protein Data Bank database (https://www1.rcsb.org/). Based on these crystal structures, the position of the cysteine mutation should not affect the function and structure of OmCI itself and should be easy to modify the subsequent albumin binding fragment, that is, it should meet the following three points: (1) conservative mutations should be selected to avoid affecting the binding of OmCI to its receptor, that is, the amino acids involved in the interaction should not be mutated; (2) the formation of three pairs of disulfide bonds should not be affected; (3) the position should be solvent accessible on the protein surface. T90 (SEQ ID NO: 4), K95 (SEQ ID NO: 5), T97 (SEQ ID NO: 6), E126 (SEQ ID NO: 7) and S156 (SEQ ID NO: 8) of OmCI were selected for cysteine mutation. These OmCI cysteine mutants were engineered, fermented and purified as described in Example 2. Except for the OmCIE126C mutant, which was difficult to express and purify, the remaining mutants were successfully prepared.

(2) OmCIT90C, OmCIK95C, OmCIT97C, OmCIE126C, and OmCIS156C were expressed in the form of tag fusion proteins. Taking OmCIT90C as an example, an expression cassette containing an N-terminal 6-histidine (His ₆ ) tag and a ubiquitin-like modifier protein (SUMO) tag for the expression of OmCI mutants was constructed and inserted into the pET30 plasmid. Then it was transformed into competent E. coli BL21 (DE3) bacteria, and an engineered strain for expressing His ₆ -SUMO-OmCIT90C (SEQ ID NO: 3) fusion protein was obtained by screening. The engineered bacteria were fermented at high density to express the His ₆ -SUMO-OmCIT90C fusion protein in a soluble form in the periplasmic space.

(3) First, purify the soluble fusion protein His ₆ -SUMO-OmCIT90C (SEQ ID NO: 3), obtain the soluble target OmCIS156C mutant protein by enzyme digestion, and then purify to obtain the soluble target OmCIT90C mutant protein with higher purity. Add 0.05% PEI to the bacterial resuspension and extract at 50°C for 2h to extract the soluble fusion protein expressed in the periplasmic space; during the entire separation and purification process, the resuspension buffer is supplemented with 10mM cysteine as a blocking agent, so that the mutant cysteine on the target protein can bind to the exogenous free cysteine to form the fusion protein His ₆ -SUMO-OmCIT90C-Cys, and prevent aggregation between protein monomers. The pH of the extract was then adjusted to 6.0, and after centrifugation, the extract reached a clear state, and then the pH was adjusted to 7.4. The fusion protein was enriched by metal ion chelation chromatography (IMAC) HisTrap Ni excel (5 ml, purchased from Cytiva), and the fusion protein containing the His ₆ tag was eluted by 0.3 M imidazole. The eluted peak was collected and subjected to anion exchange chromatography (HiTrap Q HP (5 ml, purchased from Cytiva), the chromatography pH was maintained at 7.4, and eluted with a linear salt gradient of 0-500 mM NaCl, and then ULP1 enzyme (which can specifically cut the SUMO tag) was added to the eluted peak for enzyme cleavage at 32°C, and the cleaved solution was subjected to metal ion chelation chromatography (IMAC) to remove the tag protein (His ₆ -SUMO), and the effluent was collected and combined after SDS-PAGE analysis and RP-HPLC analysis to obtain the target OmCIT90C (SEQ ID NO:4) mutant, which mainly exists in the form of the target OmCIT90C-Cys mutant protein in which the mutant cysteine is blocked, accompanied by the free monomer form (OmCIT90C) in which the cysteine is not blocked. Finally, the OmCI mutant protein was concentrated by ultrafiltration using a MWCO 3kDa filter membrane and replaced in PBS.

After LC-MS analysis, the measured molecular weight of the monomer OmCIT90C without blocked cysteine prepared according to the above method was 16781.2Da, which was consistent with the theoretical molecular weight (16781.63Da). The measured molecular weight of the cysteine-blocked OmCIT90C-Cys mutant was 16900.80Da, which was consistent with the theoretical molecular weight (16900.79Da).

In the following examples, the remaining OmCI mutant proteins were expressed in a bacterial expression system such as E. coli BL21 (DE3). Unless otherwise specified, the expression and purification were performed according to the method described in this example. The results are as follows:

Table 1. LC-MS analysis results of OmCI protein and its mutants

Example 3: Synthesis of albumin binding fragments

3.1 Synthesis of CM03:HO ₂ C-(CH ₂ ) ₁₆ -CO-AEEA-γGlu-γGlu-Lys-CO-CH ₂ -Br

Synthesize brominated fatty acid side chains according to solid phase chemistry: weigh 1.00 g of 2-CTC resin, place it in a peptide synthesizer reactor, add 10 ml of DCM, swell for 1 hour, weigh 2-3 times the amount of Fmoc-Lys(Alloc)-OH and absorb 4-6 times the amount of DIEA, add 10 ml of DMF to dissolve, put it into the reactor, react at room temperature for 2 hours, that is, the first amino acid is coupled to the resin, then wash the resin with DCM 6 times, and measure the substitution value (SD) of the resin at this time; then add 10 ml of 20% PIP/DMF solution, mix for 10 minutes to remove the amino protecting group Fmoc, repeat this process once, then wash the resin with DCM 6 times, couple the second amino acid, weigh three times the amount of Fmoc-Glu-otBu, HOBt and DIC, add 10 ml of DMF/DCM (1:1) mixed solvent to dissolve, react at room temperature, monitor the reaction progress with ninhydrin, and the reaction is complete when it is colorless, and wash the resin with DCM 6 times. Then, the reaction can be continued according to the above coupling method, and this cycle is repeated until the coupling is completed.

Weigh tetrakis(triphenylphosphine)palladium (0.1 times the amount) and phenylsilane (10 times the amount), add 15 ml DCM to dissolve, and then add to the reactor to react with the resin peptide for 25 minutes. This process must be carried out under light-proof and nitrogen protection conditions. After the reaction is completed, use DCM (15 ml*6 times, 2 minutes each time), 0.02 mol/L N, N-diethyldithiocarbamic acid/DMF solution (15 ml*3 times, 2 minutes each time), DMF (15 ml*6 times, 2 minutes each time) and take a small amount of resin for ninhydrin detection. The purple-black color of the resin particles indicates that the deprotection is complete.

Add 2 equivalents of bromoacetyl bromide and 10 ml of DMF, react at room temperature for 1 h, monitor the reaction progress with ninhydrin, and If the color turns red, the reaction is complete, wash the resin with DCM for 6 times, and dry the resin peptide in vacuum for later use.

The cleavage reagent was added in the ratio of 1g resin to 10ml cleavage reagent, the reagent ratio was TFA:TIS: _H2O =95:2.5:2.5 (V:V), the reaction was carried out at room temperature for 1h, the resin was removed by filtration, the filtrate was rotary evaporated at 40°C to remove TFA as much as possible, the fatty acid side chain was precipitated with 7-10 times ice ether, the fatty acid side chain was placed in a -20°C refrigerator for 20min, centrifuged, and vacuum dried to obtain the fatty acid side chain.

Theoretical molecular weight: 966.96Da; measured molecular weight: 967.43Da.

In the following examples, the solid phase chemical synthesis of brominated fatty acid side chains and other albumin binding fragments will be carried out according to the method described in this example unless otherwise specified.

3.2 Synthesis of CM04:HO ₂ C-(CH ₂ ) ₁₄ -CO-AEEA-γGlu-γGlu-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 938.91Da; measured molecular weight: 939.40Da.

3.3 CM05: Synthesis of HO ₂ C-(CH ₂ ) ₁₈ -CO-AEEA-γGlu-γGlu-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 995.02Da; measured molecular weight: 995.55Da.

3.4 CM06: Synthesis of HO ₂ C-(CH ₂ ) ₂₀ -CO-AEEA-γGlu-γGlu-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 1023.07Da; measured molecular weight: 1023.60Da.

3.5 CM07: Synthesis of HO ₂ C-(CH ₂ ) ₁₄ -CO-γGlu-AEEA-AEEA-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 954.95Da; measured molecular weight: 955.44Da.

3.6 CM08: Synthesis of HO ₂ C-(CH ₂ ) ₁₆ -CO-γGlu-AEEA-AEEA-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 983.01 Da; measured molecular weight: 983.49 Da.

3.7 CM09: Synthesis of CH ₃ -(CH ₂ ) ₁₆ -CO-γGlu-AEEA-AEEA-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 953.02Da; measured molecular weight: 953.30Da.

3.8 Synthesis of CM10:HO ₂ C-(CH ₂ ) ₁₈ -CO-γGlu-AEEA-AEEA-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 1011.06Da; measured molecular weight: 1011.51Da.

3.9 Synthesis of CM11:HO ₂ C-(CH ₂ ) ₁₄ -CO-γGlu-εLys-εLys-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 920.99 Da; measured molecular weight: 921.49 Da.

3.10 Synthesis of CM12:HO ₂ C-(CH ₂ ) ₁₆ -CO-γGlu-εLys-εLys-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 949.04Da; measured molecular weight: 949.53Da.

3.11 Synthesis of CM14:HO ₂ C-(CH ₂ ) ₁₈ -CO-γGlu-εLys-εLys-Lys-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 977.09 Da; measured molecular weight: 977.59 Da.

3.12 CM15: Synthesis of [HO ₂ C-(CH ₂ ) ₁₆ -CO-γGlu-AEEA-AEEA-Lys-CO-] ₂ -C ₆ H ₃ -NH-CO-CH ₂ -Br

Procedure: Prepare as described in 3.1.

Theoretical molecular weight: 1988.00Da; Measured molecular weight: 1989.68Da

Example 4: Preparation of OmCI mutant derivatives modified with albumin binding fragments

4.1 Preparation of OmCIT90C-CM03: OmCI-Cys ⁹⁰ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₁₆ -CO ₂ H]

The compound is an OmCIT90C mutant derivative (OmCIT90C-CM03) modified by the albumin binder (CM03) as described in Example 3.1, such as the OmCIT90C mutant of SEQ ID NO:4 (Example 2). The specific preparation process and identification are as follows.

Procedure: Before the modification reaction, the cysteine-blocked OmCIT90C-Cys mutant was ultrafiltered into PBS buffer (pH 7.4) with a final concentration of 1 mg/mL. At the same time, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine, dissolved in PBS buffer, adjusted to pH 7.4) was added and reacted in a water bath at 37°C for 2 h. The albumin binder as described in Example 3.1 was dissolved in a saturated ammonium bicarbonate solution, and the dissolved albumin binding fragment solution was added to the OmCI mutant at a molar ratio of 1:10 for OmCI mutant:albumin binding fragment, and reacted overnight under stirring. After the reaction, the albumin binder-modified OmCI mutant derivative was purified by anion exchange chromatography 30Q column (purchased from Suzhou Saifen). The chromatography pH was maintained at 7.4, and eluted with a linear salt gradient of 0–500 mM NaCl. The target peaks were merged after RP-HPLC analysis and SDS-PAGE analysis to obtain the OmCIT90C mutant of SEQ ID NO: 4 with higher purity (Example 2), which was an OmCI mutant derivative modified with the albumin binding fragment (CM03) as described in Example 3.1.

Theoretical molecular weight: 17667.68Da; measured molecular weight: 17667.12Da.

4.2 Preparation of OmCIT90C-CM05: OmCI-Cys ⁹⁰ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIT90C mutant of SEQ ID NO:4 (Example 2) and the albumin binding fragment (CM05) as described in Example 3.3. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17695.68Da; measured molecular weight: 17694.83Da.

4.3 Preparation of OmCIT90C-CM06: OmCI-Cys ⁹⁰ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₂₀ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIT90C mutant of SEQ ID NO:4 (Example 2) and the albumin binding fragment (CM06) as described in Example 3.4. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17725.68Da; measured molecular weight: 17724.12Da.

4.4 Preparation of OmCIT90C-CM10:OmCI-Cys ⁹⁰ -[S-CH ₂ CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative obtained by modifying the OmCIT90C mutant of SEQ ID NO: 4 (Example 2) with an albumin binder (CM10) as described in Example 3.8. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17711.78Da; measured molecular weight: 17711.29Da.

4.5 Preparation of OmCIT90C-CM14:OmCI-Cys ⁹⁰ -[S-CH ₂ -CO-Lys-εLys-εLys-γGlu-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIT90C mutant of SEQ ID NO:4 (Example 2) and the albumin binder (CM14) as described in Example 3.11. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17677.81Da; measured molecular weight: 17677.10Da.

4.6 Preparation of OmCIT97C-CM03:OmCI-Cys ⁹⁷ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₁₆ -CO ₂ H]

The compound is an OmCI mutant derivative modified with an albumin binder (CM03) as described in Example 3.1 by the OmCIT97C mutant of SEQ ID NO:6 (Example 2), and the preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17667.68Da; measured molecular weight: 17668.24Da.

4.7 Preparation of OmCIT97C-CM05: OmCI-Cys ⁹⁷ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is the OmCIT97C mutant of SEQ ID NO: 6 (Example 2) obtained by mixing the white The preparation process and identification of the OmCI mutant derivative modified with the protein binding fragment (CM05) are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17695.68Da; measured molecular weight: 17695.25Da.

4.8 Preparation of OmCIT97C-CM06: OmCI-Cys ⁹⁷ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₂₀ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binding fragment (CM06) as described in Example 3.4. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17725.68Da; measured molecular weight: 17724.36Da.

4.9 Preparation of OmCIT97C-CM09:OmCI-Cys ⁹⁷ -[S-CH ₂ CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₆ -CH ₃ ]

The compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binder (CM09) as described in Example 3.7. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17653.74Da; measured molecular weight: 17651.22Da.

4.10 Preparation of OmCIT97C-CM10:OmCI-Cys ⁹⁷ -[S-CH ₂ CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binder (CM10) as described in Example 3.8. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17711.78Da; measured molecular weight: 17711.26Da.

4.11 Preparation of OmCIT97C-CM14:OmCI-Cys ⁹⁷ -[S-CH ₂ -CO-Lys-εLys-εLys-γGlu-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIT97C mutant of SEQ ID NO:6 (Example 2) and the albumin binder (CM14) as described in Example 3.11. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17677.81Da; measured molecular weight: 17677.28Da.

4.12 Preparation of OmCIS156C-CM03: OmCI-Cys ¹⁵⁶ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₁₆ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM03) as described in Example 3.1. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17681.70Da; measured molecular weight: 17681.64Da.

4.13 Preparation of OmCIS156C-CM05: OmCI-Cys ¹⁵⁶ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binding fragment (CM05) as described in Example 3.3. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17709.70Da; measured molecular weight: 17709.74Da.

4.14 Preparation of OmCIS156C-CM06: OmCI-Cys ¹⁵⁶ -[S-CH ₂ CO-Lys-γGlu-γGlu-AEEA-CO-(CH ₂ ) ₂₀ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binding fragment (CM06) as described in Example 3.4. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17737.70Da; measured molecular weight: 17737.80Da.

4.15 Preparation of OmCIS156C-CM07: OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₄ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binding fragment (CM07) as described in Example 3.5. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17669.69 Da; measured molecular weight: 17669.28 Da.

4.16 Preparation of OmCIS156C-CM08: OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₆ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM08) as described in Example 3.6. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17697.75Da; measured molecular weight: 17697.34Da.

4.17 Preparation of OmCIS156C-CM09: OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₆ -CH ₃ ]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM09) as described in Example 3.7. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17667.76Da; measured molecular weight: 17667.39Da.

4.18 Preparation of OmCIS156C-CM10: OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM10) as described in Example 3.8. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17725.80Da; measured molecular weight: 17725.24Da.

4.19 Preparation of OmCIS156C-CM11: OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-εLys-εLys-γGlu-CO-(CH ₂ ) ₁₄ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM11) as described in Example 3.9. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17635.73Da; measured molecular weight: 17636.43Da.

4.20 Preparation of OmCIS156C-CM12:OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-εLys-εLys-γGlu-CO-(CH ₂ ) ₁₆ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM12) as described in Example 3.10. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17663.78Da; measured molecular weight: 17662.74Da.

4.21 Preparation of OmCIS156C-CM14:OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-Lys-εLys-εLys-γGlu-CO-(CH ₂ ) ₁₈ -CO ₂ H]

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM14) as described in Example 3.11. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 17691.83Da; measured molecular weight: 17691.31Da.

4.22 OmCIS156C-CM15:OmCI-Cys ¹⁵⁶ -[S-CH ₂ -CO-NH-C ₆ H ₃ -[-CO-Lys-AEEA-AEEA-γGlu-CO-(CH ₂ ) ₁₆ -CO ₂ H] ₂ ] Preparation

The compound is an OmCI mutant derivative modified by the OmCIS156C mutant of SEQ ID NO:8 (Example 2) and the albumin binder (CM15) as described in Example 3.12. The preparation process and identification are as follows.

Procedure: Prepare as described in 4.1.

Theoretical molecular weight: 18702.74Da; measured molecular weight: 18702.19Da.

Table 2. LC-MS analysis results of OmCI modified derivatives

Example 5: Classical complement pathway hemolysis test (CH50)

For the classical pathway hemolysis inhibition assay (CH50), the test was performed according to the method described in CN1798841B. 2 mL of fresh sterile sheep defibrinated blood in Alsever's (1:1 v/v) was washed once with 20 mL of GVBE (0.1% gelatin, 5 mM barbital, 145 mM NaCl, 10 mM EDTA; pH 7.4) buffer, centrifuged at 2500 x g for 10 min, the supernatant was discarded, and then washed three times with 20 mL of GVB++ (0.1% gelatin, 5 mM barbital, 145 mM NaCl, 0.15 mM CaCl ₂ , 0.5 mM MgCl ₂ ; pH 7.4) buffer. The precipitated red blood cells were diluted in GVB++ buffer in proportion to prepare 4×10 ⁸ cell/mL of sheep erythrocytes were reacted with 2 units (1:4000) of rabbit hemolysin (purchased from Shanghai Yuanye) in a 1:1 ratio and incubated in a 37°C water bath for 30 min to prepare 2x10 ⁸ cell/mL sensitized sheep erythrocytes (EA) (antigen-antibody immune complex). In the reaction system, 95uL of normal human serum NHS (final concentration 2% v/v)/guinea pig serum GPS (final concentration 0.5% v/v) diluted in GVB++ buffer, purchased from Beijing Borsi) was used as a complement source, and 10uL of different concentrations (0.2nM–500nM) of the test sample/PBSE (10mM EDTA; positive control, set to 100% inhibition)/PBS (negative control, set to 0% inhibition) was added, and then mixed with 95uL sensitized sheep red blood cells (EA). At the same time, 95uL EA, 95uL water and 10uL PBS were mixed as a 100% hemolysis control, incubated at 37°C for 0.5h, centrifuged at 2500xg for 10min, and 100uL of the supernatant was added to a 96-well plate to measure its absorbance at 412nm (A _412nm ). Thus, the amount of hemoglobin released during the lysis of red blood cells was quantified. The experiment was repeated three times independently, and the inhibition rate of the sample was calculated according to the hemolysis rate. Finally, the half-maximal inhibitory concentration (IC50) value of the test sample was obtained by four-parameter fitting.
Inhibition rate % = 1 - hemolysis rate % ((A _412nm sample - _{A 412nm} positive) / (A _412nm negative - _{A 412nm} positive))

Example 6: Alternative complement pathway hemolysis test (AH50)

The alternative pathway hemolysis inhibition assay (AH50) was similar to the CH50 and tested according to the method described in CN1798841B. 2 mL of sterile rabbit defibrinated blood in Alsever's was washed three times with 20 mL of GVBMg-EGTA buffer (0.1% gelatin, 5 mM barbital, 145 mM NaCl, 2.5 mM MgCl ₂ , 8 mM EGTA; pH 7.4). The precipitated red blood cells were diluted in GVBMg-EGTA buffer in proportion to prepare 2×10 ⁸ cell/mL of rabbit red blood cells. In the reaction system, 95uL of normal human serum NHS (final concentration 43%) diluted in GVBMg-EGTA buffer was used as a complement source, and 10uL of different concentrations (0.6nM-1500nM) of the test sample/test sample/PBSE (10mM EDTA; positive control, set to 100% inhibition)/PBS (negative control, set to 0% inhibition) was added, and then mixed with 95uL RE, and water was used as a 100% hemolysis control. Incubate at 37°C for 0.5h, centrifuge at 2500xg for 10min, take 100uL of the supernatant and add it to a 96-well plate, and measure its absorbance at 412nm. The experiment was repeated three times independently. The inhibition rate of the sample was calculated according to the hemolysis and lysis rate, and finally the half-maximal inhibition concentration (IC50) value of the test sample was obtained by four-parameter fitting.
Inhibition rate % = 1 - hemolysis rate % ((A _412nm sample - _{A 412nm} positive) / (A _412nm negative -
A _412nm positive)

Example 7: In vitro anti-complement classical/alternative pathway activity (CH50/AH50) of OmCI recombinant protein, OmCI mutants and their modified derivatives

The in vitro activities of recombinant OmCI protein, cysteine-blocked OmCI mutants and modified derivatives thereof (compounds in Examples 1, 2 and 4) were evaluated by the methods of Examples 5 and 6. The results are shown in Table 3. The successfully expressed recombinant OmCI protein and its mutants all have considerable in vitro complement hemolytic inhibition activity, and fatty acid modification does not affect the complement inhibition activity of OmCI.

Table 3. In vitro complement classical/alternative pathway hemolysis inhibition results of OmCI mutants and their derivatives

Example 8: Evaluation of complement hemolysis inhibition activity (PNH50) using PNH-like erythrocytes

Inhibition of complement alternative pathway-mediated erythrocyte lysis was evaluated using an in vitro model of PNH-like erythrocytes. Normal human erythrocytes were sensitized with 2-aminoethylisothiourea bromide (AET) to inactivate complement regulatory proteins on the cell surface and induce a PNH-like phenotype as follows:

2 mL of normal human erythrocytes were mixed with 2 mL of Aldrich's solution, washed three times with PBS, centrifuged at 2500 x g, and 10% of the upper layer of erythrocytes was removed each time. 1 mL of the washed erythrocytes was incubated with 4 mL of 8% w/v AET (pH 8.0) at 37°C for 10 min for sensitization, and then washed three times with PBS. Thereafter, the concentration of erythrocytes in the erythrocyte stock solution was adjusted with PBS so that the absorbance at 405 nm was 1.5-2.0 (indicating hemoglobin release) when 10 μL of the erythrocyte stock solution was diluted with 140 μL of water.

1.5 mL of the above erythrocyte stock solution was mixed with anti-CD55 antibody (BRIC216; Sigma-Aldrich, final concentration 6.7 μg/mL) and incubated on ice for 30 minutes. After washing once with PBS, the treated erythrocytes were resuspended in PBS-Mg (1 mM MgCl ₂ pH 6.4) to an initial volume of 1.5 mL. This erythrocyte preparation with induced PNH phenotype was subjected to the following AP-mediated lysis assay.

Normal human serum was acidified to pH 6.4 with 0.2M HCl and supplemented with MgCl ₂ and EGTA to a final concentration of 2.5mM and 8mM, respectively. 60μL normal serum NHS (final concentration of about 53% v/v) was added to the reaction system and mixed with 10μL PBS-Mg (set to 100% hemolysis as a negative control)/PBS-EDTA (10mM EDTA; set hemolysis rate to 0% as a positive control)/10μL samples of different concentrations (0.6nM-1500nM), the sample and serum were incubated on ice for 5 minutes, and then 10μL sensitized red blood cells were added to supplement 20μL PBS-Mg buffer. Incubate at 37°C at 900rpm for 30 minutes, and then quench the reaction by adding 50μL ice-cold PBS containing 10mM EDTA. The remaining unlysed cells were centrifuged at 2500xg for 10 min, and 100uL of the supernatant was added to a 96-well plate to measure its absorbance at 405nm ( _A405nm ). The amount of hemoglobin released during the lysis of red blood cells was quantified. The experiment was repeated three times independently, and the inhibition rate of the sample was calculated based on the hemolysis rate. Finally, the half-maximal inhibition concentration ( _IC50 ) value of the test sample was obtained by four-parameter fitting.
Inhibition rate % = 1 - hemolysis rate % ((A _405nm sample - _{A 405nm} positive) / (A _405nm negative -
A _405nm positive)

The recombinant OmCI protein (Example 1), the OmCI modified derivative OmCIT90C-CM05 (Example 4.2) and OmCIT90C-CM14 (Example 4.5) was evaluated for complement inhibition activity using PNH-like erythrocytes. The results are shown in Table 4, and the successfully expressed recombinant OmCI protein and its modified derivatives have considerable in vitro complement hemolytic inhibition activity.

Table 4. Results of in vitro PNH-like erythrocyte hemolysis inhibition by OmCI and its derivatives

Example 9. Molecular level receptor protein complement C5 binding study

The affinity between the recombinant OmCI protein (Example 1), the mutant OmCIT90C (Example 2), the OmCI modified derivative OmCIT90C-CM05 (Example 4.2) and OmCIT90C-CM14 (Example 4.5) and the receptor C5 was determined using surface plasmon resonance (SPR) technology. Real-time affinity measurements were performed at 25°C on a Biacore 8000 system, using PBST-0.02 buffer as a running buffer and a blank control, and the samples to be tested were gradiently diluted using the running buffer to a concentration of 300nM-3nM. The receptor protein (human complement C5 protein) was diluted to a concentration of 10μg/mL with a 10mM sodium acetate (pH 5.0) solution, and the human complement C5 protein was fixed on a CM5 sensor chip using an amino coupling method in PBS buffer at a continuous flow rate of 10μL/min and 200s, and its ligand density was approximately 1900-2400 resonance units (RU). The experiment was carried out in PBST-0.02 running buffer with different concentrations of the sample dilution and blank control, and flowed through the control channel and the experimental channel at a flow rate of 30 μL/min, with a binding time of 60 s and a dissociation time of 200 s. Real-time data signals were collected using BiaControl Software 2.0. 2M MgCl ₂ solution was used for regeneration at a flow rate of 30 μL/min for 30 s to interrupt the strong electrostatic interaction between OmCI protein and C5.

The average baseline determined by three injections of buffer was corrected, the corresponding signal measured by the blank channel of the online control was subtracted, the data were fitted to the Langmuir 1:1 binding model formed by the bimolecular complex, and the kinetic parameters were determined by calculation using BiaEvaluation Software 2.0 software. The results are listed in Table 5, which shows that OmCI and its mutants and fatty acid modified derivatives have an in vitro affinity equivalent to that of human complement C5 protein, and mutation or modification does not affect the binding ability of OmCI protein to receptor C5.

Table 5. Affinity of OmCI and its mutants and modified derivatives for complement C5 in vitro

Example 10. Molecular level albumin binding studies

The affinity between recombinant OmCI protein (Example 1), mutant OmCIT90C (Example 2), OmCI modified derivatives OmCIT90C-CM05 (Example 4.2) and OmCIT90C-CM14 (Example 4.5) and human serum albumin (HSA) was determined using surface plasmon resonance (SPR) technology. Real-time affinity measurements were performed at 25°C on a Biacore 8000 system (GE Healthcare). 0.02% PBST buffer was used as the running buffer and blank control, and the samples to be tested were gradiently diluted with the running buffer to a concentration of 3000nM-40nM. Human serum albumin (HSA) was diluted to a concentration of 10μg/mL with 10mM sodium acetate (pH 4.5) solution, and HSA was fixed on a CM5 sensor chip using an amino coupling method in PBS buffer at a continuous flow rate of 10μL/min and 200s, with a ligand density of approximately 3500 resonance units (RU). The experiment was carried out in the running buffer with different concentrations of the sample dilution and blank control, flowing through the control channel and the experimental channel at a flow rate of 30μL/min, with a binding time of 60s and a dissociation time of 200s. Real-time data signals were collected using BiaControl Software 2.0. 10mM Gly-HCl solution (pH 2.5) was used for regeneration at a flow rate of 30μL/min for 30s to interrupt the strong electrostatic interaction between fatty acids and albumin.

The average baseline determined by three injections of buffer was corrected, the corresponding signal measured by the blank channel of the online control was subtracted, and the data were fitted to the Langmuir 1:1 binding model of bimolecular complex formation, and the kinetic parameters were determined by calculation using BiaEvaluation Software 2.0 software. The results are listed in Table 6, which show that OmCI and its mutants do not bind to human serum albumin (HSA), while acylated OmCI protein can bind to HSA through the coupled fatty acid.

Table 6. Affinity of OmCI and its mutants and modified derivatives to human serum albumin (HSA)

Example 11. Pharmacokinetic studies

Female C57BL/6J mice aged 6-8 weeks were housed under standard conditions. Mice (approximately 20 g) were divided into three groups and given a single dose of OmCI (s.c. 15 mg/kg); OmCI-T90C-CM05 (s.c. 15 mg/kg); OmCIT90C-CM14 (s.c. 15 mg/kg) in PBS by subcutaneous injection in the neck. Blood samples were collected through the tail vein at 0.1, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after administration (three animals per group at each time point). The blood was drawn into EDTA·2K-coated tubes, placed on ice, and centrifuged at 1200×g for 10 min at 4°C. The plasma was then transferred to Micronic tubes and stored at -20°C. The target protein concentration in plasma was determined by sandwich ELISA.

A transparent 96-well plate was supplemented with 50 μL of human complement C5 protein (purchased from Complement Technology) dissolved in PBS at 10 μg/mL and coated overnight at 4°C. After washing three times with PBS containing 0.05% v/v Tween 20 (PBST-0.05), 200 μL Blocker Casein (purchased from Thermo Fisher Scientific) was used at room temperature in the blocking Block for 1 hour and wash again. The plasma samples were diluted 1:5 in PBS to a constant content of up to 20% v/v mouse plasma in the sample matrix and incubated at 37°C for 1 hour. After washing, 50 μL of rabbit polyclonal antibody (obtained by immunostimulation from Anhui Global Gene Technology) diluted to 5 μg/mL in blocking solution was added for 1 hour. After washing, 50uL of HPR-goat anti-rabbit IgG (AB205718; purchased from Abcam) diluted in 1:4000 blocking solution was added for 1 hour and color was developed by adding 100 μL TMB (purchased from Thermo Fisher Scientific) to each well. After 15 minutes, 2M sulfuric acid was added to terminate the reaction and the absorbance was measured at 450nm. A separate standard curve was constructed to determine the plasma level of each protein, which was obtained from a dilution series of purified recombinant OmCI protein with a determined concentration in PBS, also supplemented with up to 20% v/v mouse plasma from untreated animals. The plasma concentration (μg/mL) of each sample was calculated using the parameters derived from the standard curve (log-log regression analysis). The pharmacokinetic parameters were calculated using a two-compartment model and drug statistics software (DAS, version 2.0; Chinese Mathematical Pharmacology Committee). The results are listed in Table 7.

Table 7. Pharmacokinetic parameters of OmCI mutants and their derivatives in mice

Abbreviations: t1/2α distribution half-life; t1/2β elimination half-life Cmax: maximum plasma concentration; CL clearance; AUC: area under the curve;

The sequences used in this article are as follows:

SEQ ID NO:1

>Natural OmCI

SEQ ID NO:2

>OmCI protein with periplasmic binding protein and enterokinase recognition site fused to the N-terminus

SEQ ID NO:3

>OmCI mutants containing an N-terminal 6-histidine (His6) tag and a SUMO-like modifier (SUMO) tag Variant (His6-SUMO-OmCIT90C)

SEQ ID NO:4

>OmCI mutant T90C (OmCIT90C)

SEQ ID NO:5

>OmCI mutant K95C (OmCIK95C)

SEQ ID NO:6

>OmCI mutant T97C (OmCIT97C)

SEQ ID NO:7

>OmCI mutant E126C (OmCIE126C)

SEQ ID NO:8

>OmCI mutant S156C (OmCIS156C)

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

A modified protein comprises a protein portion and a modification portion, wherein a cysteine residue is introduced into the protein portion by mutation, and the modification portion is connected to the protein portion via the cysteine residue introduced into the protein portion, and the protein is OmCI or an active fragment thereof. A OmCI mutant having the amino acid sequence shown in SEQ ID NO:1, and T90, K95, T97, E126 or S156 are mutated to cysteine residues. A pharmaceutical composition comprising the modified protein according to claim 1 or the OmCI mutant according to claim 2 and a pharmaceutically acceptable excipient. Use of the modified protein according to claim 1 or the OmCI mutant according to claim 2 in the preparation of a drug. An albumin binder, the structure of which is shown in the following formula:
[R ₁ -(X ₁ ) _m -(X ₂ ) _n -K] _o -X ₃ -(ε-R ₂ ); Where K is lysine _R1 is a substituted or unsubstituted _C1-20 acyl group; m is an integer from 0 to 5; n is an integer from 0 to 5; o is an integer of 1-3 (preferably 1 or 2); _X1 and _X2 are independently selected from the following group: alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), tranexamic acid (Trx), 8-amino-3,6-dioxaoctanoic acid (AEEA), PEG; X ₃ is absent or is a linking moiety of the following formula: (R ₃ ) _o R ₄ R ₅ ; wherein R ₃ is a substituted or unsubstituted C _1-3 acyl group, R ₄ is a C _5-8 aryl or heteroaryl group, and R ₅ is a substituted or unsubstituted amino group; _R2 is selected from halogen-substituted _C1-6 acyl. An isolated nucleic acid molecule encoding the OmCI mutant of claim 2. An expression vector comprising the isolated nucleic acid molecule of claim 6. A host cell, wherein the host cell comprises the expression vector according to claim 7 or the nucleic acid molecule according to claim 6 is integrated into the genome of the host cell. Use of the OmCI mutant according to claim 2 in preparing the modified protein according to claim 1. A method of treatment, comprising the step of administering a therapeutically effective amount of the modified protein of claim 1, the OmCI mutant of claim 2, or the pharmaceutical composition of claim 3 to a subject in need thereof.